Int. J. Cancer; 65,168-172 (1996) 0 1996 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de I‘Union Internationale Contre le Cancer OVER-EXPRESSION OF HEPATOCYTE GROWTH FACTOR IN HUMAN KAPOSI’S SARCOMA Jeanette A.M. MAIER2-5,Massimo MA RIOT TI^, Adriana A L B I N I ~Paola , cOM12, Maria PRAT3, Paolo M. cOMOGLI03 and Marco R. SORIA’ Departments of ‘Biological and TechnologicalResearch-Dibit, San Raffaele Institute, Milan, 2BiomedicalSciences and Technologies HSR, University of Milan, Milan; and 3BiomedicalScience and Oncoloa, University of Turin, Turin; and 4NationalInstitute of Research on Cancer, Genoa, Italy. Kaposi’s sarcoma is a highly vascularired multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cy-tokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposi’ssarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesionderived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte-growthfactor receptor (c-MET). Moreover,elevated levels of transforming growth factor P I (TGFPI) mRNA were found in lesions of human sporadic Kaposi’s sarcoma and in lesion-derived spindle cells which also over-express urokinase. Since HGF, TGFP I and urokinase are all involved in capillary-vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposi’s sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion. o 1996 Wiley-Liss, Inc. Kaposi’s sarcoma (KS) in general, and acquired-immunodeficiency-syndrome-related KS (AIDS-KS) in particular, is an invasive and intensely angiogenic multifocal neoplasm of unknown cellular origin. It has been suggested that the multiple simultaneous KS lesions arise in response to systemic or micro-environmental alteration(s), such as increase in circulating cytokines, widespread dysregulation of the immune system, and/or multiple opportunistic infections. KS lesions contain a complex mixture of cell types, including fibroblasts, endothelial cells, dendritic cells and leukocytes of different lineages (Templeton, 1988). The presence of spindle cells is a distinctive histological marker of KS, and these cells are thought to be the proliferative component of the lesion. However, their nature and origin are highly controversial. The pathogenesis of KS is unknown, though epidemiologic features suggest a viral etiology (Chang et al., 1994). HIV itself is a co-factor in patients with AIDS-related KS, as suggested by the induction of KS-like lesions in HIV tat-transgenic mice (Corallini et al., 1993; Vogel et al., 1988). These data suggest that growth factors and the HIV Tat protein released by retrovirus-infected T lymphocytes act together to induce proliferation of KS spindle cells, as well as the activation and proliferation of endothelial cells, thus promoting angiogenesis. Hepatocyte growth factor (HGF), also known as scatter factor, is a cytokine secreted by mesenchymal cells and endowed with uncommon mitogenic and motogenic properties (Montesano et al., 1991). The specific receptor for HGF is a transmembrane heterodimeric tyrosine kinase encoded by c-met (Naldini et al., 1991). HGF primarily functions as an endocrine or paracrine mediator of epithelial-mesenchymal interaction. Endothelial cells, also, express functional HGF receptors, and HGF is a powerful angiogenic factor in vitro and in vivo (Bussolino et al., 1992). HGF and c-met are coexpressed in AIDS-related KS lesions, suggesting that HGF might play a role in the pathogenesis of AIDS-KS (Naidu et al., 1994; Polverini and Nickoloff, 1995). Because of the distinctive ability of HGF to direct biological processes leading to angiogenesis, and because KS lesions are highly vascularized, we studied the expression of HGF and its receptor in spindle cells from human sporadic or AIDS-associated KS, and in specimens from sporadic KS patients. In all cases we found an inappropriate expression of both HGF and its receptor. We also found high levels of expression of TGFp1. Moreover, cultured spindle cells synthesize higher amounts of urokinase than endothelial cells. Urokinase is implicated in the activation of the TGFpl and HGF precursors, and all these molecules stimulate angiogenesis and invasion (Mignatti and Rifkin, 1993). We argue that coordinated dysregulation of these interacting components might be relevant in the pathogenesis of KS. MATERIAL AND METHODS Cell cultures and biopsies Human umbilical-vein endothelial cells (HUVEC) were generated and maintained as described (Bussolino et al., 1992). The IST-KS2 and IST-KS3 cell lines were obtained from sporadic and from AIDS-related KS lesions respectively (Albini et al., 1992). Smooth-muscle cells were grown in D-MEM with 10% FCS. Specimens from male patients with sporadic KS lesions were obtained from Dr. G. Zambruno (Modena, Italy). Northem-blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) RNA from cell cultures and from human KS lesions were extracted by the cesium-chloride method. RNA (15 pg/lane) was resolved on 1% agarose-formaldehyde gels, and Northernblot hybridization was performed at high stringency. Human HGF, urokinase and TGFPl cDNAs were labeled with [32P] dCTP (specific activity 3000 Ci/mmol) by the random-primer technique and used as a probe. GAPDH hybridization was used to show that equal amounts of RNA were loaded. For RT-PCR, 1 pg total RNA was reverse-transcribed, the cDNA product was boiled for 5 min, and PCR amplification was carried out using 1/50 of the final R T reaction as described (Sambrooket al., 1989). Each amplification cycle consisted of 1 min denaturation at 95”C, 2 min annealing at 55°C and 2 min extension at 72°C. Thirteen cycles were performed using 30 pmoles of each primer. One fifth of the PCR reaction mix was electrophoretically separated on a 2% agarose gel containing ethidium bromide and Southern blot was performed. This procedure renders the result of RT-PCR analysis quantitative. The sequences of the HGF primers were: 5’-GAA ACT GCA TCA TTG GTA AAG GA-3’ (sense) and 5‘-TAT CAT CAA AGC CCT TGT CGG GA-3’ (anti-sense). The sequences of the human c-met primers were: 5‘-ACA GTG GCA TGT CAA CAT CGC T-3‘ (sense) and 5’-GCT CGG TAG TCT ACA 5To whom correspondence and reprint requests should be sent, at Department of Biomedical Sciences and Technologies-HSR, University of Milan, 58 Via Olgettina, 20132 Milan, Italy. Fax: +39-2-26434767. Received: July 18, 1995 and in revised form September 6,1995. HEPATOCYTE GROWTH FACTOR IN KAPOSI’S SARCOMA 169 GAT TC-3’ (anti-sense). The sequences of the TGFpl primers were: 5’-ACG TGC AAG ACT ATC GAC ATG GAG-3’ (sense) and 5’-GTTATC CCT GCT GTC ACA GGA GCA-3’ (anti-sense). The sequence of the human FGF-2 primers were: 5’-GGA GTG TGT GCT AAC CGT TAC CTG GCT ATG-3‘ (sense) and 5‘-TCA GCT CTT AGC AGA CAT TGG AAG AAA AAG-3‘ (anti-sense). Antibodies The murine monoclonal antibody (MAb) DO-24, directed against the extracellular domain of the HGF receptor, was obtained following immunization with living cells from the human gastric-carcinoma cell line GTL16. The MAb EA-7 was obtained by immunizing mice with purified recombinant human HGF. Polyclonal anti-MET antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Westem-blot analysis Conditioned media collected from human IST-KS and their corresponding controls were incubated with 100 p1 of heparinSepharose (Pharmacia, Uppsala, Sweden) overnight. HeparinSepharose was recovered by centrifugation, washed with 10 mM Tris, pH 7.5, containing 1 mM EDTA. Proteins were eluted by boiling in sample buffer without reducing agents, separated by electrophoresis in a 8% polyacrylamide gel and transferred to nitrocellulose sheets. The blot was incubated with the EA-7 MAb diluted 1:250, and then with iodinated protein A. After extensive washing, the blot was exposed to Kodak XAR film. To detect c-MET, cells were lysed in RIPA buffer and incubated with the anti-MET MAb DO-24. After 6 hr, protein G-Sepharose was added. Bound proteins were eluted by boiling in SDS sample buffer, separated by electrophoresis in a 8% polyacrylamide gel under non reducing conditions, and transferred onto a nitrocellulose membrane. The blot was incubated with the polyclonal anti-MET antibody SP-60 (Santa Cruz Biotechnology) and then visualized with alkaline-phosphatase-conjugated anti-rabbit IgG and BCIP/ NBT substrates (Promega Biotec, Madison, WI). u-PA assay Confluent IST-KS and HUVEC were lysed in Tris-HC1, pH 8.0, containing 1% Triton X-100; 10 pg of cell extract and 100 pl of culture media were used to measure u-PA, using the plasmin chromogenicsubstrate H- D- norleucyl- hexahydrotyrosyl- lysine- p- nitroanilide- acetate (American Diagnostica, Greenwich, CT), according to manufacturer’s instructions, and an automatic microplate reader at 405 nm. The experiments were performed in triplicate. RESULTS Over-expression of HGF and its receptor in human KS HGF and c-met were recently found over-expressed in AIDS-KS lesions (Naidu et al., 1994). We have extended this finding by studying the expression of HGF and c-met by RT-PCR with HGF- and met-specific primers followed by Southern hybridization, in specimens from human sporadic KS. As shown in Figure 1, HGF is up-regulated in sporadic KS lesions derived from 3 different patients, and c-met mRNA is readily detectable in the lesions as well as in normal skin. We also evaluated the expression of HGF and c-met in 2 different human spindle-cell types. One type was established from an AIDS-related KS lesion (IST-KS3), the other from a sporadic KS lesion (IST-KS2) (Albini et al., 1992).By Northernblot analysis, we could detect a band of 6 Kb hybridizing with the HGF probe only in IST-KS2 cells, whereas IST-KS3 cells, human endothelial cells derived from the umbilical cord, and human vascular smooth-muscle cells were all negative (Fig. 2a). By immunoblot analysis on conditioned media after concentration on heparin-Sepharose, IST-KS2 cells were found FIGURE1 -Expression of HGF, c-met, TGFPl and FGF-2 in skin specimens from sporadic KS. Total RNA (1 p+) extracted from skin lesions of sporadic KS was reverse-transcribed, amplified by PCR (13 cycles) using specific primers, and Southern blot was performed as described in “Material and Methods”. Lane 1, control normal skin; lane 2, lesion of sporadic KS, case 1; lane 3, lesion of sporadic KS, case 2; lane 4, lesion of sporadic KS, case 3. to secrete HGF (Fig. 2b, lane 3), whereas in endothelial cells, smooth-muscle cells and IST-KS3 cells HGF was undetectable (Fig. 2b, lanes 1 , 2 and 4). To investigate whether MET was present in these cells, lysates were immunoprecipitated with a MAb directed against the extracellular domain of c-MET. The immune complexes were then transferred to nitrocellulose under non-reducing conditions and probed with a polyclonal antibody (SP-60) against c-MET. As shown in Figure 3, a band of the expected size was readily detected in endothelial and in IST-KS cells. Expression of FGF-2 and TGFpI in human KS cells and in human lesions AIDS KS lesions have been shown to express mRNA for a complex mixture of cytokines, that might account for some of the biological properties of these cells: in particular, it has been reported that interleukin 1 (IL-1) (Y and P are overexpressed in these lesions, whereas the expression of FGF-2 is debated (Ensoli et al., 1989; Schulze-Osthoff et al., 1990). In the human IST-KS cells analyzed in this study, we found neither IL-1 (not shown) nor FGF-2 over-expression (Fig. 4). Actually, IST-KS3 cells down-regulate FGF-2 mRNA (Fig. 4). An increase in FGF-2 mRNA was consistently detected in 2 specimens from sporadic KS (Fig. 1).Finally, by Northern-blot analysis using a human TGFPl cDNA, we observed that IST-KS2 cells over-express TGFPl (Fig. 2a). Analogous results were obtained when specimens from sporadic KS patients were examined by RT-PCR analysis followed by Southern hybridization (Fig. 1). It is noteworthy that the specimen derived from patient 1was found to over-express HGF, TGFP and FGF-2. Over-expression of these 3 angiogenic factors was 170 MAIER ETAL. FIGURE2 - Expression of HGF and TGFpl in human KS-derived spindle cells and corresponding controls. (a) Total RNA (15 pg) prepared from different cell types was fractionated on 1%agarose-formaldehyde gel, and Northern-blot analysis was performed at high stringency using human HGF and TGFpl cDNA probes. Hybridization with GAPDH indicates that similar amounts of RNA were used per lane. Lane 1, smooth-muscle cells; lane 2, HUVEC; lane 3, IST-KS2; lane 4, IST-KS 3. (b) Conditioned media from different cell types were concentrated on heparin-Sepharose and used for Western-blot analysis using a MAb against human HGF, as described in “Material and Methods”. Lane 1, smooth-muscle cells; lane 2, HUVEC, lane 3, IST-KS2; lane 4, IST-KS3. FIGURE3 -1ST-KS cells express c-MET. Cell extracts were immunoprecipitated using an anti-c-MET antibody. Immunoprecipitates were resolved on an 8% SDS-PAGE in non-reducing conditions, and Western blot was performed using an anti-c-MET antibody. Lane 1, HUVEC; lane 2, IST-KS2; lane 3, IST-KS3. FIGURE4 - E ression of FGF-2 in IST-KS cells. Total RNA (1 pg) extracteTfrom IST-KS cells and HUVEC was reversetranscribed, amplified by PCR (13 cycles) using specific primers, and Southern blot was performed as described in “Material and Methods”. Lane 1, IST-KS2; lane 2, IST-KS3; lane 3, HUVEC. GAPDH amplification shows that equal amount of RNA were loaded. DISCUSSION correlated with a more aggressive form of KS (G. Zambruno, personal communication). Expression of urokinase in IST-KS cells Because both T G F p l and H G F precursor are activated to the correspondent biologically active forms by urokinase (u-PA), we evaluated the expression of this protease in IST-KS cells. Northern analysis shows that IST-KS cells over-express u-PA transcripts (Fig. 5a). This result was parallelled by the finding that IST-KS cells produce and secrete higher amounts of u-PA than endothelial cells used as a control. However, while IST-KS3 cells expressed higher amounts of uPA mRNA, the level of enzymatic activity was similar in both IST-KS cell types (Fig. 5b). This could be due to inefficient activation of pro-uPA, or to different amounts of PA inhibitor(s) in the cell environment. Kaposi’s sarcoma is a highly vascularized tumor which often complicates HIV infection. It is not fully understood which factors are involved in the onset and progression of KS, and which factors induce the impressive angiogenic reaction invariably associated with the development of KS lesions. The Tat protein of HIV stimulates the growth of KS cells in vitro (Ensoli et al., 1990), and tat transgenic mice develop KS-like lesions (Corallini et al., 1993; Vogel et al., 1988). Thus, it has been hypothesized that cytokines and Tat act synergistically in the development of KS in AIDS patients (Barillari et a[., 1992). However, other viral etiologic agents might also have an important role in the pathogenesis of KS, perhaps by perturbing cytokine interactions in a hitherto uncharacterized fashion (Chang et al., 1994). HGF is a multifunctional cytokine endowed with growthstimulatory properties o n a variety of cell types expressing the appropriate receptor (Naldini et al., 1991); among these are HEPATOCYTE GROWTH FACTOR IN KAPOSI’S SARCOMA 171 endothelial cells. The latter finding explains the powerful angiogenic activity displayed by HGF (Bussolino et al., 1992). W e here report that HGF is expressed in sporadic human KS specimens as well as in KS spindle cells obtained from human sporadic KS lesions. Moreover, IST-KS2 cells release HGF. Our data rule out any role of the Tat protein of HIV, since sporadic KS is negative for tat expression. Moreover, Tat does not induce H G F in endothelial cells of different origin (data not shown). We have also found elevated levels of T G F p l mRNA in lesions of human sporadic KS and in IST-KS spindle cells. The molecular mechanisms that up-regulate HGF and TGFP1 levels in these cells are presently unknown. Since u-PA plays a role in the activation of the latent forms of both HGF and TGFP (Mignatti and Rifkin, 1993; Naldini et al., 1992), it is noteworthy that IST-KS cells up-regulate urokinase. Thus, HGF might stimulate production of urokinase (Pepper et al., 1992), which is in turn implicated in the activation of the precursor. Interestingly, u-PA is a hallmark of neoplastic transformation (Mignatti and Rifkin, 1993), and Lunardi-Iskandar et al. (1995) have provided evidence that KS can evolve into a malignancy. O u r results show that IST-KS2 cells express not only HGF, but also the HGF receptor c-met. It is worth noting that, in epithelial cells, the co-expression of HGF and c-met activates the transforming activity of the receptor (Rong et al., 1992) and that an HGF autocrine loop leads to tumorigenesis (Bellusci et al., 1994). In this perspective, H G F may initiate and maintain an autocrine loop that stimulates growth of KS spindle cells, playing a critical role in the pathogenesis of Kaposi’s sarcoma. 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