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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.
Moreover, since HGF induces blood-vessel formation in vivo
and stimulates endothelial-cell migration and proliferation in
vitro (Bussolino et al., 1992), its secretion by KS cells may
cooperate to produce the relevant angiogenesis observed in
Kaposi’s sarcoma.
2a
b
HUVEC
IST-KSZ
IST-KS3
FIGURE
5 - Expression of u-PA in human KS-derived spindle
cells. (a) Total RNA (15 pg/lane) prepared from different cell
types was fractionated on an agarose-formaldehyde gel as described in “Material and Methods” and hybridized with a human
u-PA cDNA probe. Lane 1, smooth-muscle cells; lane 2, HUVEC;
lane 3, IST-KS2; lane 4, IST-KS3. Hybridization with GAPDH
is shown in Figure 2. (b) Cell-associated and secreted u-PA
were measured using a chromogenic assay. Data are shown as the
mean 2 standard deviation of 2 separate experiments performed
in triplicate.
ACKNOWLEDGEMENTS
We are grateful to Dr. G. Barbanti-Brodano for critical
reading of the manuscript, and to Dr. F. Blasi and Dr. G.
Consalez for helpful discussions. This research was supported
by grants to J.A.M.M. from Associazione Italiana per la
Ricerca sul Cancro (AIRC), and to M.R.S. from Progetto
AIDS 1994-1995, Minister0 della Saniti, Rome; Consiglio
Nazionale delle Ricerche Progetto Finalizzato ACRO; AIRC;
and Regione Lombardia.
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