Induction of Hepatocyte Growth Factor in Fibroblasts by Tumor-Derived Factors Affects Invasive Growth of Tumor Cells: in Vitro Analysis of Tmnor-Stromal Interactions'

Home , Fibroblast growth factor, Hepatocyte growth factor

[CANCERRESEARCH57,3305-3313,August1, 1997] Induction of Hepatocyte Growth Factor in Fibroblasts by Tumor-derived Factors Affects Invasive Growth of Tumor Cells: In Vitro Analysis of Tmnor-Stromal Interactions'

Takao Nakamura, Kunio Matsumoto, Akira Kiritoshi, Yasuo Tano, and Toshikazu Nakamura2

Division of Biochemistry, Biomedical Research Center [Ta. N., K. M., To. N.J and Department of Ophthalmology [A. K., Y. T.J, Osaka University Medical School, Suita, Osaka 565, Japan

ABSTRACT and metastasis. In addition to this stromal involvement in tumor malignancy, several studies have shown that stromal alterations pre Invasive and metastatic potentials of several types of carcinoma cells cede the malignant progression of tumor cells (3, 5), including the are regulated through interactions with host stromal cells, e.g., tumor activated appearance of stromal fibroblasts surrounding the tumor stromal interactions. Because hepatocyte growth factor (HGF), a ligand for the c-Met proto-oncogene product, Is a mesenchymal- or stromal cells. Therefore, local and mutual interactions between carcinoma derived factor that induces mitogenic, motogenic, and morphogenic re cells and stromal fibroblasts are of particular importance in regulating sponses, we examined the mechanisms involved in tumor-stromal inter the extracellular matrix degradation, migration, and invasion of tumor actions in vitro. The c-Met/HGF receptor was expressed in A431 human cells. The molecular mechanisms leading to tumor progression are epidermoid carcinoma cells, A549 human non-small cell lung cancer cells, unclear; hence, molecular mechanisms for tumor-stromal interactions HuCC-T1 human cholangiocellular carcinoma cells, and SBC-3 human that confer the malignant characteristics of tumor cells are of interest. small cell lung carcinoma cells. HGF stimulated cell growth, scattering, HGF,3 initially identified and cloned as a potent mitogen for mature and invasion of these cells. Although these cells did not produce biologi hepatocytes (6â€”8),is a mesenchymal (stromal)-derived multipotent cally significant levels of HGF, these cells did secrete soluble factors that growth factor that elicits mitogenic, motogenic, and glandular mor potently stimulated HGF production in human skin fibroblasts. These phogenic activities on various types of cells, mostly in a paracrine carcinoma cell-derived HGF inducers proved to be interleukin-1 (IL-i) in A431cells,IL-i plusbasiclibroblastgrowthfactor (bFGF)in A549and fashion (9â€”13).HGF is also known to promote angiogenesis in vivo HuCC-T1 cells, and bFGF plus platelet-derived growth factor in SBC-3 and in vitro (14, 15). The HGF receptor is a membrane-spanning cells. When these carcinoma cells were cocultured with fibroblasts, HGF heterodimeric tyrosine kinase, c-Met, and it is predominantly cx levels in the coculture system were much higher than the levels in fibro pressed in various types of normal and malignant cells (16, 17). blasts alone, without cocultured carcinoma cells. Together with the in Functional coupling between HGF and c-Met mediates epithelial crease in HGF levels, the number of invasive cells increased, but in vitro mesenchymal interactions for the development of several organs. invasion of carcinoma cells in the coculture system was strongly inhibited Mice embryos deficient in HGF are lethal due to impaired develop by anti-HGF antibodies. Thus, there are mutual interactions between ment of the placenta and liver (18, 19). in vitro studies indicate that carcinoma cells and fibroblasts: IL-i, bFGF, and platelet-derived growth HGF supports morphogenesis of the kidney, mammary gland, and factor derived from tumor cells play a role in inducing HGF expression in tooth (10â€”12,20). Although interactions between the epithelium and stromal fibroblasts, whereas fibroblast-derived HGF, in turn, leads to invasive growth in carcinoma cells. The mutual interactions, as medinted mesenchyme mediate crucial aspects of normal development, these by HGF and HGF inducers, may play a significant role in the occurrence interactions may also be important in neoplasia. Molecular mecha of invasion and metastasis of carcinoma cells. nisms underlying epithelial mesenchymal interactions are likely to be functioning, at least in part, in interactions between epithelial tumors, i.e. , carcinoma cells and surrounding stromal cells. HGF stimulates INTRODUCTION the cell migration and motility of severaltypes of carcinomacells, An essential characteristic of malignant cancer cells is their poten eventually promoting progression to a more malignant phenotype tial to proliferate and infiltrate surrounding normal tissue. In most (21). cancers, lethality is the result of local invasion and the metastasis of We now report that HGF is a predominant fibroblast-derived factor neoplastic cells from the primary tumor to other tissues. Most human that stimulates mitogenesis, motogenesis, and the invasion of human malignant tumors are carcinomas, and several lines of studies done in carcinoma cells. Moreover, the mutual interactions between carci vivo (1) and in vitro (2, 3) indicated that the growth and invasive noma cells and fibroblasts are of significant importance; carcinoma potentials of carcinoma cells are influenced through interactions with cells secrete various types of HGF inducers for fibroblasts. These host stromal cells. in vivo growth of certain carcinoma cells was mutual interactions, as mediated by HGF and the inducers, may play markedly accelerated by a broad spectrum of fibroblasts, and in vitro a significant role in the occurrence of invasion and metastasis of invasion of oral squamous carcinoma cells was induced by coculti carcinoma cells. vation with stromal fibroblasts (4). A soluble factor that stimulates the invasive growth of tumor cells was partially purified from the condi MATERIALS AND METHODS tioned media of fibroblasts (3). Thus, host stromal-derived factor is likely to be one of the key molecules that regulates tumor cell invasion Materials. Human recombinant HGF was purified from the culture media of Chinese hamster ovary cells transfected with an expression plasmid con mining human HGF cDNA, and the purity of HGF exceeded 98% (8, 22). Received 12113/96;accepted 6/2/97. The costs of publication of this article were defrayed in part by the payment of page Human recombinant bFGF was a kind gift from Dr. K. Nishikawa (Kanazawa charges. This article must therefore be hereby marked advertisement in accordance with Medical College, Kanazawa, Japan). Bovine aFGF and human recombinant 18 U.S.C. Section 1734 solely to indicate this fact. FGF-4 (HST-1IFGF-4) were obtained from Toyobo Co. (Osaka, Japan) and I This study was supported by a research grant for science and cancer research from the Ministry and Education, Science, Sports and Culture of Japan, a research grant for cancer research from the Ministry of Welfare of Japan, and research grants from the Yasuda 3 The abbreviations used are: HGF, hepatocyte growth factor; IL, interleukin; IL-1R, Medical Foundation, Kudo Foundation, and Tanabe Medical Frontier Foundation. IL-i receptor; FGF, fibroblast growth factor; bFGF. basic FGF; PDGF, platelet-derived 2 To whom requests for reprints should be addressed. Phone: 81-6-879-3783; Fax: growth factor; aFGF, acidic FGF; KOF, keratinocyte growth factor; EGF-R, epidermal 81-6-879-3789. growth factor receptor; RT-PCR, reverse transcription-PCR. 3305

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Fig. I . Effects of HGF on cell growth and DNA synthesis of HOF(nglml) HGF(nglml) various carcinoma cell lines. Effects on cell growth (â€¢)and DNA synthesis (0) were presented as the fold increase relative to that of unstimulated control cells. Data, mean Â±SD of triplicate measure ments. I I @ 0 S 016SBC-3 2A S II U U .E C 11.1 â€˜2 1.3-h 1.7 .2

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R&D Systems (Minneapolis, MN), respectively. HumanrecombinantKGF of 10â€•cells/cm2on 24-well plastic plates (Costar Corp.) and cultured for an was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Human additional 24 h. The medium was changed to serum-free medium, and the cells recombinant IL-1f3and human recombinant PDCIFwere obtained from Gen were cultured for 24 h. The cells were stimulated with various concentrations zyme Corp. (Boston, MA). Polyclonal antibody against human HGF was of HGF for 15h and then pulse-labeled with 0.3 MCi/miâ€˜25Ilabeleddeoxyuri prepared from the serum of a rabbit immunized with human recombinant HGF, dine (2200 Ci/mmol; DuPont New England Nuclear, Boston, MA) for 12 h. and antihuman HGF IgG (I @g/ml)completely neutralized the biological The radioactivity incorporated into nuclei was counted using a gamma counter. activities of 1 ng/ml human HGF. Polyclonal antibody against the human The stimulatory effect on cell motility (cell scattering) was measured as c-Met/HGF receptor (C-l2) was obtained from Santa Cruz Biotechnology described elsewhere (23). (Santa Cruz, CA). Monoclonal mouse antihuman EGF-R antibody (a-EGF-R) In Vitro Invasion Assay. In vitro invasion of carcinomacells was meas was obtained from Genzyme Corp. Monoclonal mouse antihuman bFGF ured using Biocoat Matrigel invasion chambers (Collaborative Biomedical antibody (a-bFGF) was a kind gift from Dr. K. Nishikawa. Recombinant Products, Bedford, MA). The cells were seeded on Matrigel invasion chamber human IL-lR antagonist and polyclonal goat antihuman PDGF antibody (a plates at a density of5 X l0@cells/cm2and cultured in DMEM containing 10% PDGF) were obtained from R&D Systems and Promega (Madison, WI), FCS. HGF was added to the lower medium, and cells were cultured for 3 days. respectively. Invasive cells that penetrated through pores and migrated to the underside of Assay for Cell Growth, DNA Synthesis, and Cell Motility. A431 human the membrane were stained with H&E and counted with microscopic vision. epidermoid carcinoma cells, A549 human non-small cell lung cancer cells, For cocultivation of carcinoma cells and fibroblasts, human dermal fibre HuCC-Tl human cholangiocellular carcinoma cells, and SBC-3 human small blasts were initially seeded on 24-well plates at a density of 5 X 10â€•cells/cm2 cell lung carcinoma cells were obtained from the Japanese Cancer Research and cultured in DMEM containing 10% FCS for 24 h. The medium was Resources Bank. Normal human skin fibroblasts were initially proliferated changed to fresh medium supplemented with 2% FCS, and carcinoma cells outward from dermal tissue obtained during plastic surgery. These cells were were seeded on the inner cup of 24-well Matrigel invasion chamber plates at cultured in DMEM supplemented with 10% FCS. a density of 5 X l0@cells/cm2 and cultured for 3 days (A43l, A549, and To measure cell growth, 10â€•cellswere seeded on 6-well plates (Costar HuCC-Tl cells) or 7 days (SBC-3 cells). Fifty percent (v/v) ofthe medium was Corp., Cambridge, MA) and cultured for 24 h. After washing with DMEM, the changed every 3 days. cells were cultured for 7 days in DMEM supplemented with 1% FCS (A549 Measurement of HGF In CUltUre Media To prepare conditioned media and SBC-3 cells) or 2% FCS (A431 and HuCC-T1 cells) in the absence or from carcinoma cells, confluent carcinoma cells were washed three times and presence of various concentrations of HGF. The medium was changed every 3 incubated in DMEM supplemented with 1% FCS for 3 days. Human fibre days. The number of cells was counted using a hemocytometer after dissoci blasts were seeded on 48-well plates at a density of 5 X 10@'cells/cm2and ation by trypsin. To measure DNA synthesis, the cells were seeded at a density cultured for 24 h. After replacing the media with fresh DMEM supplemented 3306

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Fig. 2. Cell scattering induced by HGF. Cells were plated at a density of I X 10' cells/well (A431 and @@@@ HuCC-T1 cells), 1 X 10@cells/well (A549 cells), or . - câ€¢Ãª 4 X lO@cells/well (SBC-3 cells) on 6-well plates and HuCC-T1 cultured for 24 h in the absence or presence of 5 ng/ml is./,'.â€” . @(, ii,,, @ HGF. Bar, 200 ,xm. â€¢.

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@ with 1% FCS and 2 @xg/mlheparin,cytokines or conditioned media from 20 - -@ - carcinoma cells were added, and the cells were cultured for 24 h. The concentration of HGF in the media was determined by ELISA, as described ,@. @:A431 @ elsewhere (24). :2 :A549 @ Western Immunoblotting of HGF. HGF in the conditioned medium from i@ 15 :HuCC-T1 @ :@ :SBC-3 @ fibroblasts was partially purified using Hi-Trap heparin (PharmacialBiotech) 0 10 and subjected to SDS-PAGE using a 4â€”20%gradient gel under nonreducing @a conditions, as described elsewhere (25). After SDS-PAGE, the proteins were .u, w electrophoretically transferred onto a polyvinylidene difluoride membrane U @@@ (Bio-Rad, Hercules, CA). Immunoblotting was done as described elsewhere 01 .â€˜.â€˜ (25). Western Immunoblotting of the c-Met/HGF Receptor. After reaching confluence, carcinoma cells were scraped, and cells were collected by centrif @;5 i@ ugation and then solubilized in 500 @lofice-coldlysis buffer composed of 150 mM NaCl, 20 mM Tris-HCI (pH 7.5), 10 mM EDTA, 5 @xg/mlleupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.5% (v/v) Triton X-lOO. After centrifu gation, supematants were incubated with antibodies against the c-MetIHGF @@ receptor for 1 h at 4Â°C,andimmune complexes were recovered with protein I :@h@ 20 A-Sepharose 4FF (Pharmacia/Biotech). After SDS-PAGE under reducing con ditions, the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad), and the membrane was incubated with antibodies against the c-MetJHGF receptor. The membrane was incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Inc., Burlingame, CA) and subsequently HGF (ng/mI) incubated with peroxidase-conjugated avidin-biotin complex and then visual Fig. 3. Enhancement by HGF of cell invasion through Matrigel membrane. The cells ized using a chemiluminescent kit (Amersham, Bucks., United Kingdom). were seeded on 24-well Matrigel invasion chamber plates at a density of 5 X l0@cells/cm2 Analysis of iasI@labeled HGF Binding to the Receptor. Recombinant and cultured in DMEM containing 10% FCS. HGF was added to the lower media, and human HGF was radioiodinated by the chloramine-T method, and concentra cells were cultured for 3 days. Values, mean Â±SD of triplicate measurements. 3307

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C m 0 0 0 50 100 0 50 100 Fig. 4. Expression of c-Met/HGF receptor. A, con centration-dependent 25l-labeled HGF binding to its 125 l4gfl@(p@) 125 @44tF(p@) receptor on carcinoma cells. inset, a Scatchard plot. Kd and B,,@ values were calculated from the Scatchard 20 15 analysis. B, expression of c-MetIHGF receptor immu noprecipitated from the cell lysate of carcinoma cells. 0 Cell lysate from cultured carcinoma cells was immu a @ noprecipitated with antibodies against the c-Met/HGF 15 receptor and subjected to SDS-PAGE under reducing C' 10 conditions. The proteins were transferred and probed with antibodies against the c-Met/HGF receptor. C, expression of c-Met mRNA in carcinoma cells detected by RT-PCR. RT-PCR was carried out using specific 5 primers for the c-MetIHGF receptor or @-actingene I (see â€œMaterialsandMethodsâ€•).PCR products were separated on 2% agarose and stained with ethidium 0 I bromide. Arrowhead, the product of the expected 0 0 length of 0 50 100 0 50 100 516 bp. 125 @4$GF(pM) 125 I-HGF(pM)

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tion-dependent binding of â€˜251-labeled HGF to cells was analyzed as described I I elsewhere (23). c-Met/HGF Receptor mRNA Expression. c-Met mRNA expression was analyzed by RT-PCR. RNA was purified from cultured carcinoma cells using 6 the acid guanidinium thiocyanate-phenol chloroform method (26). The primers used for PCR were as follows: 5'-GmCCCAA1TFCI@GACC-3' and 5'- TATATCAAAGGTG1TFAC GT-3', the c-MetIHGF receptor; and 5'-CGT GAAAAGATGACCCAGAT-3' and 5'-ATT GCCGATAGTGATGACCT-3', the @3-actingene.Amplification was performed for 35 cycles (30 s at 94Â°C,1 4 mm at 52Â°C,and 1.5 mm at 72Â°C).PCR products were separated on 2% agarose and stained with ethidium bromide. Northern Blot Hybrldlzatlon RNA was purified from cultured cells using the acid guanidimum thiocyanate-phenol chloroform method (26), and North em blot hybridization was done as described elsewhere (24). 2

RESULTS

Mitogenic and Motogemc Activities and Enhancement of in Vitro Invasion. We first examined the effects of HGF on prolifera I I I tion and DNA synthesis of A43l, A549, HuCC-T1, and SBC-3 cells â€œ0 -25 50 (Fig. 1). HGF dose-dependently stimulated cell growth and DNA CM (% In v/v) synthesis of the four cell lines, and the maximal stimulatory effects Fig. 5. HGF-inducing activity in conditioned media of various carcinoma cells. Human seen with 10â€”30ng/ml HGF were 1.3-, 1.6-, 1.5-, and 2.2-fold in fibroblasts were seeded on 48-well plates and cultured for 24 h. After replacing the A43l, A549, HuCC-Tl, and SBC-3 cells, respectively. This means medium with fresh DMEM supplemented with I% FCS and 2 pg/mi heparmn,appropriate amounts of conditioned medium from carcinoma cells were added, and the cells were that HGF has weak mitogenic activity for A43l, A549, HuCC-Tl, and cultured for 24 h. The concentration of HGF in the medium was measured by ELISA. SBC-3 carcinoma cells. Values, mean Â±SD of triplicate measurements. 3308

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Fig. 6. Induction of HOF by FGFs, PDGF, and IL-l. A, induction of HGF mRNA expression. Human fibroblasts were cultured in the absence or presence of FGFs (10 ng/ml), lL-l@ (0.25 ng/ml), or PDGF (10 ng/ml) for 12 h, and RNA was purified from the cells. Total RNA (30 @g)waselectrophoresed, and RNA blots were hybridized with 32P-labeled human HGF cDNA. rRNAs stained with ethidium bromide are shown in the lowerphotograph to indicate the amount of RNAs loaded onto the gel. B. stimulation of HGF production in human fibroblasts by FGFs, PDGF, and IL-l$. Human fibroblasts were seeded on 48-well plates and cultured for 24 h. After replacing the medium with fresh DMEM supplemented with 1% FCS and 2 @.tWmiheparmn,cells were cultured in the absence or presence of FGFs (10 ng/ml), 1L-l(3 (0.25 ng/ml), or PDGF (10 ng/ml) for 24 h. Values, mean Â±SD of triplicate measurements. C, Western inununoblotting of HGF produced by human fibroblasts. Human fibroblasts were cultured in the absence or presence of 10 ng/ml FGFs for 48 h. HGF in the conditioned medium was partially purified and subjected to SDS-PAGE. After electroblotting. HGF was probed with rabbit antihuman HGF lgCi.Thefirst lane shows recombinant human HGF as an authentic sample.

To determine whether HGF would stimulate cell motility, these cate that HGF is potent in stimulating the motility and invasion of carcinoma cells were cultured in monolayer, and HGF was added these carcinoma cells. (Fig. 2). HGF caused a dissociation of the colonies and stimulated Expression of the c-Met/HGF Receptor. Because the above re motility, resulting in scatterings of A43l, A549, and SBC-3 cells. sults indicate that A43l, A549, HuCC-Tl, and SBC-3 carcinoma cells Although HGF did not cause a remarkable dissociation of colonies in are targets of HGF, we analyzed the expression of c-Met in cultured HuCC-Tl cells, the cells had a flattened and spindle-like appearance carcinoma cells. Fig. 4A shows the concentration-dependent binding when HGF was added, and several cells were scattered, thereby of â€˜25I-labeledHGF to carcinoma cells in culture. â€˜251-labeledHGF suggesting that HGF stimulated motility. Thus, HGF has motogenic specifically bound to these cells with Kd values between 31â€”36pM, activity for A43l, A549, HuCC-Tl, and SBC-3 cells, whereas and the number of high-affinity binding sites/cell was calculated as HuCC-Tl seems to form more adhesive cell-cell interactions than do 348 sites/cellinA431, 324 sites/cellinA549, 1685sites/cellin A431, A549, and SBC-3 cells. HuCC-Tl, and I 14 sites/cell in SBC-3 cells, respectively (Fig. 4A). We next measured the invasion of carcinoma cells in vitro using a We also detected c-Met/HGF receptor expression by Western im Matrigel invasion chamber. In the absence of HGF, there was no munoblotting (Fig. 4B). The f3 subunit (Mr 145,000; p 145 f3) was evidence of invasive cells. However, HGF potently stimulated the identified in four carcinoma cell lines, and the precursor form of invasion of A431, A549, and SBC-3 cells but less potently stimulated c-Met/HGF receptor (Mr 170,000; Pr 170) was identified in A549 and the invasion of HuCC-Tl cells (Fig. 3). Again, in this case, the lesser HuCC-Tl cells (Fig. 4B). The amount of c-MetJHGF receptor de invasive potential in HuCC-Tl cells might be due to the more adhe tected by immunoblotting was relatively correlated with the number sive characteristics of these cells. Nevertheless, these results do mdi of high-affinity binding sites detected by Scatchard analysis. Consist 3309

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Fig. 7. Neutralization of HGF-inducing activity in conditioned media of various carcinoma cells. Human fibroblasts were seeded on 48-well plates and cultured for 24 h. After replacing the medium with fresh DMEM supplemented with 1% FCS and 2 @sg/mlhepaiin, conditioned medium from carcinoma cells was added in the absence or presence of antibodies, an antagonist, and their combinations. Condi tioned media were added to cultures (in v/v) as follows: A431, 6.25%; HuCC-T1 SBC4 A549, 50%; HuCC-Tl, 25%; and SBC-3, 50%. Antibodies and an 125 @â€˜125 antagonistwere addedto culturesas follows:a-EGF-R,3 @xg/ml; IL-1R antagonist, 300 ng/ml; a-bFGF, 6 pg/mi; and a-PDGF, 30 @xg/tnl.Values,mean Â±SD of triplicate measurements. 1100 @100

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ent with these results, the expression of c-Met/I-IGF receptor mRNA these carcinoma cell lines secrete soluble factors that stimulate HGF was confirmed by RT-PCR (Fig. 4C). The PCR products with the production in stromal fibroblasts. expected size of 516 bp were detected in these cells, and their amounts Induction of HGF Expression in Fibroblasts by FGF, PDGF, correlated relatively with the number of high-affinity HGF receptors and IL-i. Before determining which molecule is responsible for the on these cells. induction of HGF in fibroblasts as a carcinoma-derived factor, we Enhancement of HGF Production by Carcinoma-derived Solu examined the effects of various growth factors and cytokines on the ble Factor. During malignant progression through tumor-stromal in production of HGF in fibroblasts. As reported previously, aFGF, teractions, carcinoma cells have significant effects on stromal fibre bFGF, HST- 1IFGF-4, IL- 1, PDGF, epidermal growth factor, trans blasts, because the existence of activated fibroblasts was noted in forming growth factor a, and prostaglandins stimulated HGF produc stromal tissues associated with tumor tissues (27, 28). We then asked tion in human fibroblasts (24, 25, 29, 30). Fig. 6A shows HOF mRNA whether HGF production in stromal fibroblasts would be affected by expression in fibroblasts upon exposure to aFOF, bFGF, HST-l/ factors derived from carcinoma cells. Normal human fibroblasts were FGF-4, KGF, IL-l@3, or PDGF. HGF mRNA expression was not cultured in the presence of conditioned media derived from carcinoma detectable or only slightly detectable in the control culture, whereas cells, and HGF concentration in the culture medium was measured aFGF, bFGF, HST-l/FGF-4, IL-lf3, or PDGF did induce HGF mRNA (Fig. 5). The addition of conditioned media from A431 cells potently expression, but KGF did not do so. Consistent with the induction of stimulated HGF production, and a 9-fold stimulation occurred. Like HGF mRNA, HGF production in fibroblasts was stimulated by aFGF, wise, HGF production in fibroblasts was stimulated by conditioned bFGF, HST-IIFGF-4, IL-1f3, and PDGF but not by KGF (Fig. 6B). media from A549, HuCC-Tl, and SBC-3 cells. Maximal stimulatory Western immunoblotting analysis indicated that the addition of effects seen within the tested concentration range were 7-, 5-, and aFGF or bFGF stimulated the production of the native type of HGF 11-fold in A549, HuCC-Tl, and SBC-3 cells, respectively. Therefore, (Mr 85,000), but not the two-kringle variant form of HGF (Fig. 6C). 3310

A IL-l and PDGF also stimulated the native type of HGF production (data not shown). Native-type HGF is an 85-kDa heterodimeric mol CarcinorI cells eculecomposedofthefourkringle-containingca-chainandaserine protease-like @3-chain,whereas the 28-kDa smaller form of HGF containing two kringle domains is a naturally occurring variant. 8pm pore filter Identification of Carcinoma-derived HGF Inducers. We next @-@ - @uu.â€”I flbr@blasts studied whether carcinoma-derived HGF-inducing soluble factors are known growth factors or cytokines tested above. We used specific antibodies or antagonist for the ligand or receptor (Fig. 7). Among the B antibodies and antagonist tested, stimulatory activity for HGF pro duction in the conditioned medium of A43 1 cells was almost com 15 pletely abrogated by an IL-lR antagonist; however, it was hardly inhibited by antibodies against EGF-R, bFGF, and PDGF. For HGF inducing activity in conditioned medium of A549 cells, a-bFGF and an IL-lR antagonist showed partial inhibition, and a marginal inhib itory effect was seen with a-EGF-R and a-PDGF. Simultaneous addition of a-bFGF and an IL-lR antagonist mostly inhibited HGF inducing activity in the A549 cells. In the case of HuCC-Tl cells, an IL-1R antagonist strongly inhibited HGF-inducing activity in the conditioned medium, whereas a-bFGF partially inhibited it. Simulta neous addition of an IL-1R antagonist and a-bFGF almost completely inhibited HGF-inducing activity in HuCC-Tl cells. For SBC-3 cells, a-bFGF and a-PDGF inhibited HGF-inducing activity in a condi tioned medium of SBC-3 cells, and their simultaneous addition almost completely inhibited it. Thus, HGF-inducing factors in conditioned media proved to be as follows: IL-i in A431 cells; IL-l and bFGF in A549andHuCC-Tlcells;andbFGFandPDGFinSBC-3cells. Induction of in Vitro Invasion of Carcinoma Cells by Fibro =â€˜ 7.5 blast-derived HGF. Based on our findings that carcinoma cells se crete HGF-inducers for fibroblasts, whereas HGF potently stimulated Â£5 migration and invasiveness of carcinoma cells, we set up a coculture U- method in which fibroblasts were cultured in the lower well, whereas @2.5 carcinoma cells were cultured in the Matrigel invasion chamber (Fig. 8A). We then measured invasion of carcinoma cells and HGF pro 0 duction (Fig. 8, B and C). A431, HuCC-Tl, and SBC-3 cells did not produce detectable levels of HGF, whereas A549 cells produced 0.31 ng/ml HGF during 3 days of culture, and bFGF, IL-l , and PDGF had no effect on HGF levels in these cancer cells (data not shown). On the C other hand, without cocultured carcinoma cells, basal HGF production in fibroblasts was 0.31 ng/ml during the culture period of 3 days. :2 However, the HGF level was stimulated 26â€”28-foldby cocultivation .2 with A43l cells (Fig. 8, B and C), indicating that the A43 1-derived 10 HGF inducer (IL-l) stimulated HGF production in cocultured fibro blasts. In accordance with the increase in HGF concentration, the .1 number of invading A431 cells increased 3.5-fold by cocultivation with fibroblasts than it did without fibroblasts (Fig. 8B). Importantly, in vitro invasion of A43 1 cells was suppressed to the basal level seen without cocultured fibroblasts in the presence of anti-HGF antibody (Fig. 8, B and C). Likewise, IL-1R antagonist also inhibited the invasion of the cells, concomitant with suppression of the HGF level in this system (Fig. 8C). Similar results were obtained with the other carcinoma cell lines

=â€˜ 12 Fig. 8. In vitro invasion assay of carcinoma cells cocultured with fibroblasts. A, diagram for coculture method of carcinoma cells and fibroblasts. Human dermal fibro t blasts were initially seeded on 24-well plates, and carcinoma cells were seeded on the inner cup of 24-well Matrigel invasion chamber plates (for details, see â€œMaterialsand U- C, Methodsâ€•).Cellswere cultured for 3 days (A43l, A549, and HuCC-Tl cells) or 7 days I (SBC-3 cells). B and C, in vitro invasion and HGF levels in coculture system. Antibodies and an antagonist were added to cultures as follows: preimmune IgG, 10 pg/mi; a-HGF IgG, 10 @sg/ml;IL-lR antagonist, 300 ng/mI; and a-bFGF, 6 @sg/ml.Thecells that 0 migrated to the lower face of the membrane were counted microscopically. HGF levels were measured by ELISA. Values, mean Â±SD of triplicate measurements. W, without fibroblast; U, co-culture; â€¢,co-culture with cs-HGF lgG; 0, co-culture with IL-IR antagonist;D , co-culture with cr-bFGF;â€¢,co-culture with IL-1R antagonist + a-bFGF. 3311

tested. Cocultivation with A549, HuCC-Tl, and SBC-3 cells in carcinoma cells, which contributes to the promotion of cell motility creased HGF levels, and the number of invading carcinoma cells (39). HGF induces the expression of the urokinase plasminogen was increased by cocultivation with fibroblasts (Fig. 8B). The activator and the urokinase plasminogen activator receptor (40), addition of anti-HGF antibody into the coculture system potently which then activate the proteinase cascade that promotes focal deg inhibited invasion of A549, HuCC-Tl, and SBC-3 cells (Fig. 8B). radation of the extracellular matrix. Ligand-dependent activation of In the case of A549 cells, the number of invading cells in the c-Met in a leiomyosarcoma cell line induced a urokinase-related presence of anti-HGF antibody was significantly lower than that proteolytic network and concomitantly enhanced tumorigenicity, in without fibroblasts. This suggests that a low amount of HGF vasion, and metastasis (41). HGF, which regulates cell spreading and produced by A549 cells may affect the invasiveness of the cells in focal adhesion formation by the induction of tyrosine phosphorylation an autocrine manner. However, the neutralizing antibodies and offocal adhesion kinase (p125FAK), eventually stimulates cell motility IL- 1R antagonist had no effect on the invasion of carcinoma cells and invasion (31). Likewise, HGF activates small GTP-binding pro in the absence of fibroblasts (data not shown). Moreover, in teins, by which modulation of cytoskeleton reorganization and mem coculture system with HuCC-T1 and fibroblasts, a-bFGF and brane ruffling eventually stimulate cell motility (42). Therefore, be IL-1R antagonist inhibited the invasion of the cells, concomitant cause enhanced cell motility by HGF is coupled with the dissociation with the suppression of HGF levels (Fig. 8C). These findings in the of cell-cell interactions and extracellular matrix degradation, HGF coculture system suggest that the fibroblast-derived invasion factor seems to be particularly potent in enhancing the invasiveness of tumor for these cell lines is HGF and that HGF production in normal cells. human dermal fibroblasts is strongly stimulated by a carcinoma Cell motility factors have been implicated to play a role in stimu derived soluble factor. lating the neoplastic progression of cancer cells. Distinct types of autocrine motility factors that enhance cell motility in an autocrine DISCUSSION manner have been noted in several tumor cell lines (43), and some growth factors seem to stimulate tumor cell motility in an autocrine or Studies done during the past several years suggest that HGF is at a paracrine manner, including transforming growth factor f3 (44). On least one stromal-derived factor that may affect the motility of epi the other hand, the potent activity of HGF on epithelial cell motility thelial cells, including various types of carcinoma cells. HGF stimu has been well demonstrated; it predominantly acts as a paracrine lates the migration of various types of carcinoma cells, and factor. Nearly 90% of human malignant tumors arise from epitheial Matsumoto et a!. (31) recently identified the fibroblast-derived inva tissue, and most carcinoma cells express the c-MetIHGF receptor; sion factor for oral squamous carcinoma cells as HGF. In the present however, only a few carcinoma cells among more than 50 distinct cell study, we obtained evidence that HGF is the predominant fibroblast lines produce any detectable level of HGF.4 Therefore, most carci derived factor that promotes the mitogenesis, motogenesis, and inva noma cells are likely to use HGF derived from stromal tissue, and sion of human carcinoma cell lines A43l, A549, HuCC-T1, and importantly, carcinoma cells capable of inducing HGF production in SBC-3. Of importance in this study, we demonstrated that four car stromal cells may be particularly susceptible to a more malignant cinoma cell lines secrete inducing factors for HGF production in progression through tumor-stromal interactions. Nevertheless, a few stromal fibroblasts, and we identified the carcinoma-derived HGF types of carcinoma cells do use HGF as an autocrine motility factor, inducers as bFGF, IL-i, and PDGF. Thus, there is a mutual interaction including lung and bladder carcinoma (45, 46). between carcinoma cells and stromal fibroblasts mediated by carci HGFisa mesenchymal-orstromal-derivedparacrinemediatorthat noma-derived HGF inducers and stromal-derived HGF, which even regulates cell growth, cell motility, and morphogenesis during orga tually stimulates the migration and invasion of carcinoma cells. Other nogenesis and organ regeneration. Our present study suggests the workers also noted the presence and characterization of tumor-derived mutual interaction between carcinoma cells and stromal fibroblasts, inducing molecules for HGF (32, 33). We previously showed that mediated by carcinoma (epithelial)-derived HGF inducers and stromal human gallbladder carcinoma cells secrete IL-l as an inducer of HGF (mesenchymal)-derived HGF. We hypothesize that such mutual inter production in fibroblasts and that fibroblast-derived HGF induces action may exist in several types of tumor tissues and may explain invasion of the cells into collagen gels (34). how tumor cells acquire more malignant phenotypes, depending on We identified the carcinoma cell-derived HGF inducers to be IL-l, stromal tissues. The mutual interaction of tumor (carcinoma) cells and bFGF, and PDGF. Expression of IL-l, bFGF, and PDGF was noted in stromal fibroblasts is reminiscent of epitheial-mesenchymal (or -stro several types of carcinoma cells, and these factors may possibly mal) interactions during dynamic tissue organization and reconstruc regulate the growth of cells in an autocrine manner. The physiological tion. It may well be that several types of carcinoma cells use the significance of these factors for their own growth and their involve overlapping mechanisms of epithelial-mesenchymal interactions dur ment in invasive growth are not clear (35â€”37).Our present data ing embryogenesis or tissue regeneration, but in the case of carci indicate that IL-l, bFGF, and PDGF secreted from these carcinoma noma, it results in tumor progression or extracellular matrix remod cells are likely to play a role in inducing HGF production in certain cling. Our ongoing study is directed toward the prevention of tumor types of stromal fibroblasts, which in turn results in the acquisition of invasion and metastasis by interfering with mutual interactions be invasive growth in these carcinoma cells through the influence of tween tumor and stromal cells as mediated by HGF inducers and stromal-derived HGF. In this context, it is noteworthy that the expres HGF. sion of stromelysin 3 in stromal fibroblasts is also potently induced by growth factors, including bFGF, PDGF, and epidermal growth factor (38), similar to that of HGF. Taken together, it is probable that these ACKNOWLEDGMENTS factors derived from tumor cells activate neighboring fibroblasts, which in turn secrete proteins responsible for extracellular matrix We are grateful to Dr. K. Nishikawa for kindly providing monoclonal degradation and concomitant cell movements. mouse antihuman bFGF antibody (a-bFGF) and to M. Ohara for helpful Proteolytic degradation of the extracellular matrix, disruption of comments. cell adhesion, and cell motility are key components for tumor cell invasion. HGF induces tyrosmne phosphorylation of @3-cateninon 4 Unpublished results. 3312

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1997 American Association for Cancer Research. Induction of Hepatocyte Growth Factor in Fibroblasts by Tumor-derived Factors Affects Invasive Growth of Tumor Cells: In Vitro Analysis of Tumor-Stromal Interactions

Takao Nakamura, Kunio Matsumoto, Akira Kiritoshi, et al.

Cancer Res 1997;57:3305-3313.

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