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Tumor Biology |
Departments of Pathology [Z. S., W. C. P., N. K., P. R-B.], and Biochemistry and Molecular Biology [N. K., P. R-B.], University of Southern California, Keck School of Medicine, Los Angeles, California 90033, and Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado 80262 [A. v. B., G. J. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of the four possible isoforms, three (FGF8a, FGF8b, and FGF8e) were cloned in our laboratory from a human prostate tumor cell line, DU145 (20) . The protein products of these cDNAs share extensive amino acid homology with mouse FGF8 isoforms in that FGF8a and FGF8b exhibit identical amino acid sequences to those of their murine counterparts. The human FGF8 isoforms, although weakly expressed in human adult tissues or cell lines, are nevertheless differentially expressed. FGF8b appears to be the primary species in prostatic epithelial cell lines (20) . Consistent with previous reports, FGF8b, but not FGF8a or FGF8e, confers robust transforming and tumorigenic activities in NIH3T3 cells. This oncogenic activity becomes more relevant because evidence points to a significant up-regulation of FGF8b expression in high-grade prostate carcinomas (21 , 22) . A high frequency of FGF8 overexpression, which is associated with decreased patient survival and persists in androgen-independent disease, was detected by immunohistochemical analyses of prostate cancer specimens (22) . Furthermore, targeted overexpression of this isoform in the mammary glands of FGF8b transgenic mice results in mammary tumorigenesis (23) . In cultured human prostate cancer cells, expression of antisense FGF8b reduces their growth rate, inhibits their soft agar clonogenic activity, and decreases in vivo tumorigenicity (24) . The above findings strongly suggest that FGF8b is involved in hormone-related carcinogenesis of the prostate and mammary glands.
These observations have prompted us to investigate the biological effects of overexpression of FGF8b in prostatic cancer cells. A lentiviral transfer vector was developed for transduction of the FGF8b gene along with a GFP marker gene into the weakly tumorigenic LNCaP prostate carcinoma cell line. The proliferation, anchorage-independent growth ability, and in vitro and in vivo invasion ability of transduced LNCaP cells were determined. To examine the epithelial-stromal interactions under the condition of FGF8b overexpression in LNCaP cells, we used a coculture system in which GFP expression served as a marker for the separate quantitation of the cellular components.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8.71), and VSV
env-coding plasmids were originally obtained from Dr.
Luigi Naldini (University of Torino, Torino, Italy; Ref.
25
). The transducing vector was modified to include an
IRES-GFP marker gene cassette. Primary explant cultures of
prostate and SV stromal cells were established from residual portions
of radical prostatectomy specimens or suprapubic prostatectomy
specimens after fresh pathological inspection. The tissues were freed
of connective tissue elements by sharp dissection and minced into small
pieces (approximately 1 mm3
) using crossed No. 11
scalpel blades. They were first plated in a minimal volume of DMEM
supplemented with 20% FBS and 1% penicillin/streptomycin and were
incubated in a humidified atmosphere of 5% CO2
at 37°C. After primary outgrowth, the medium was changed to RPMI 1640
supplemented with 10% FBS. When the stromal cells had reached 70–80%
confluency, they were detached using 0.25% trypsin/1
mM EDTA and replated at 20% densities.
Construction of Transducing Vector and Generation of
Lentivirions.
Human FGF8b cDNA (20)
, which was flanked with
the CMV immediate early promoter at the 5' end and IRES-GFP
at the 3' end, was incorporated into the polycloning sites of the
transducing vector. Plasmids were amplified in Escherichia
coli and purified by Qiagen Maxi Prep kit. Using Superfect reagent
(Stratagene, Inc.), human 293T cells, at about 80% confluency, were
cotransfected with the transducing vector, packaging construct and VSV
env-encoding plasmids at a ratio of 5:5:1. The transducing
vector construct containing only the CMV-driven IRES-GFP
cassette, without FGF8b, was used in parallel cotransfection
to produce the control vector. The medium containing virions was
harvested daily starting from 3rd day to the 5th day after
transfection.
Infection of LNCaP Cells and Sorting by FACS.
The lentivirus-containing medium was concentrated 10-fold by using
Mr 300,000 molecular weight
cutoff spin columns (Gelman). One ml of concentrated
CMV-FGF8b-GFP or CMV-GFP vector lentiviral medium
was applied to 80%-confluency LNCaP cells in T25 culture flasks. After
4 h of incubation, lentiviral medium was removed. Cells were
washed with PBS twice and grown in complete medium for 2 days. The 8b-
and vector-LNCaP cells were released from flasks by trypsinization and
sorted by FACS on the basis of expression of GFP.
Northern Blot Analyses.
Total RNAs were extracted from 8b-, vector-, and noninfected LNCaP
cells by using RNeasy Mini Kit (Qiagen). RNAs were separated by
electrophoresis on a 1% denaturing formaldehyde agarose gel, and
transferred to Hybond N membrane (Amersham Corp.). The blots were
hybridized to a 32P-labeled full-length FGF8b
cDNA probe and exposed to X-ray film.
NIH3T3 Cell Transformation Assay.
To produce CM for use in the transformation assay, fresh medium without
FBS plus 10 µg/ml heparan sulfate was applied to transduced LNCaP
cells when they were at 80% confluency. After 48–72 h of incubation,
CM was collected and cell debris was removed by centrifugation. The CM,
diluted 1:10 in DMEM medium, was added to NIH3T3 cells when they had
grown to 70% confluency. After 2 days of incubation, cell morphology
was examined microscopically as described previously (20
, 24)
.
Soft Agar Clonogenicity Assay.
The 8b-, vector- and noninfected LNCaP cells were released by
trysinization and suspended in 0.4% Seakem agarose at a cell density
of 1 x 104/2 ml. The suspensions
were overlayed on 4 ml of 0.8% Seakem agarose in a 6-cm-diameter dish
and incubated at 37°C in 5% CO2 for 21 days.
The visible colonies were counted. The colony-forming efficiency was
calculated by dividing the number of soft agar colonies by the number
of cells plated and multiplying by 100 to convert to a percentage.
Matrigel Invasion Chamber Assay.
Inserts of 8-µm pore-sized membranes for 24-well plates were
prepared by coating with Matrigel basement membrane matrix (Becton
Dickinson Labware, Bedford, MA) following the manufacturer’s
instructions. Each chamber was separated into an upper and a lower
portion by the insertion of a thin layer of Matrigel basement
membrane matrix. The 8b-, vector-, or noninfected LNCaP cells were
placed on the upper chamber at a cell density of 1 x 105 cells/insert. The CM obtained by incubating
NIH3T3 cells for 24 h in serum-free DMEM in the presence of 50
µg/ml ascorbic acid was added to the lower chamber to serve as a
chemoattractant. After 24 h of incubation, the upper surface of
the inserts was wiped with cotton swabs, and the inserts were stained
with H&E. Cells that migrated through the Matrigel and the filter pores
to the lower surface were counted under a light microscope with five
random high-power fields per insert (26)
.
In Vivo Tumorigenicity and Immunohistochemistry
Studies.
The 8b-, vector-, and noninfected LNCaP cells were grown with complete
medium in log phase and released from flasks by trypsinization. One
million cells of each type were injected i.p. into athymic nude mice as
described previously (26
, 27)
. Each cell type was injected
into three animals. After 9 weeks of incubation, mice were sacrificed.
Diaphragms were fixed in 10% formalin overnight and embedded with
paraffin. Sections were incubated with a purified goat polyclonal
anti-GFP antibody (Santa Cruz) at 4 µg/ml concentration. The bound
antibody was detected with biotinylated antigoat immunoglobulin.
Sections incubated without primary antibody served as negative
controls.
Proliferation Assay of LNCaP-Stromal Coculture.
Approximately 1 x 104 8b- or
vector-LNCaP cells and 1 x 104
stromal cells were mixed and seeded into six-well plates (Corning),
whereas 1 x 104 8b-, vector-,
noninfected LNCaP cells and stromal cells alone were also plated as
controls. Each sample was seeded in triplicate. Total cell numbers of
cocultures were counted at different time points. After counting, 8b-
or vector-LNCaP cell and stromal cell cocultures were analyzed by flow
cytometry on the basis of GFP expression. The fractions of transduced
LNCaP or stromal cells were measured and multiplied by total cell
numbers to calculate the exact number for each cell type in cocultures.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ref. 25
). LNCaP cells, responsive to rFGF8b treatment and
only weakly tumorigenic in vivo, were infected with these
virions, which carried either the FGF8b-GFP or
only the GFP control gene. After infection and propagation,
the cells were sorted twice by FACS on the basis of their GFP
expression. Thus two types of populations, cells transduced with the
human FGF8b gene (designated as 8b-LNCaP) and cells
transduced with vector control construct (vector-LNCaP), were
established. Under a fluorescent microscope, GFP was an excellent
visible marker to determine whether the cells were indeed transduced
(Fig. 2A)
. The expression of FGF8b in 8b-LNCaP cells was
readily detected with a Northern blot assay using a
32P-labeled full-length FGF8b probe, whereas the
expression in vector- or noninfected cells was too low to detect by the
Northern technique (Fig. 2B)
. To demonstrate
FGF8b expression at the functional protein level, we
conducted an NIH3T3 cell biological transformation assay using CM from
transduced LNCaP cells. In agreement with our previous work
(20)
, CM from 8b-cells displayed a strong ability to
transform the NIH3T3 cells morphologically, whereas CM from
vector-LNCaP cells did not (data not shown).
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. Clearly the overexpression of FGF8b in 8b-LNCaP cells
increased the growth rate in comparison with that of vector-LNCaP
cells. After 14 days of culture, the proliferation rate of 8b-LNCaP
cells was 2-fold higher than that of vector-LNCaP cells. There was no
difference in the growth rate between vector- and noninfected LNCaP
cells (data not shown). Next, we determined whether FGF8b could
function as a mitogen to stromal cells. Two different human primary
prostatic stromal cell cultures and one primary stromal cell culture
from human seminal vesicle were used to examine the effect of rFGF8b on
stromal cell growth. Cells were treated with rFGF8b at concentrations
of 10 ng/ml or 100 ng/ml. Each experiment was repeated twice. It was
found that rFGF8b had no mitogenic effect on either prostatic (Fig. 3B)
or non-prostatic stromal cells (data not shown). In
contrast, rFGF8b, consistent with a previous study (17)
,
was able to stimulate proliferation of LNCaP cells. The stimulation
noted with 10 ng/ml of the protein factor, however, could not be
further enhanced by increasing the concentration by 10-fold (Fig. 3C)
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Next, the in vitro invasion ability of transduced cells was
measured with the Matrigel invasion chamber assay. After 24 h of
incubation, vector- (Fig. 4A)
or 8b-LNCaP cells (Fig. 4B)
that migrated
through the Matrigel basement membrane matrix and the filter pores to
the lower surface were counted by light microscopy. From 1 x 105 seeded cells, 583 ± 37.47 8b-LNCaP cells were counted compared with 173 ± 60.70 vector-LNCaP cells (Fig. 4C)
. Although the difference
between these two cell types was significant (P = 0.001), there was no significant difference between vector- or
noninfected LNCaP cells. This assay was repeated three times with cells
at different passages, and each cell sample was done in triplicate.
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. Twelve different sections for each individual diaphragm
were examined for tumor growth or tumor invasion under a microscope
(26
, 27)
. The fact that the attached cells were indeed
tumor cells was confirmed by immunohistochemistry assay using an
anti-GFP antibody (Fig. 5B)
. The immunohistochemistry assay
was repeated twice on the sections from each diaphragm. Although the
analysis involved a limited number of tissue sections, it was found
that at least four of five animals in 8b-LNCaP cell-injected mice were
positive for tumor growth, whereas all five mice in vector- or
noninfected LNCaP cell-injected mice were negative (Table 1)
. In two of these four diaphragms from 8b-LNCaP cell-injected mice,
tumor cells also exhibited multifocal attached growth on the peritoneal
surface of diaphragm. The sizes of areas that the tumor cells occupied
in these diaphragms varied considerably. In 20% of areas examined,
moreover, tumor cells did grow into the diaphragm and spread
horizontally with evidence of early vertical penetration. The latter
was characterized by tumor cells invading between muscle cells.
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. The same two human primary prostatic stromal cell
cultures described above were also used in these analyses. The stromal
cells in coculture spread out on the bottom of the culture flask
providing a mesenchymal carpet, and LNCaP cells grew on the top of
stromal cells forming gland-like structures. A microscopic view of this
coculture is presented in Fig. 6B
. The LNCaP cells were
rounder and much smaller when they were grown alone than their
counterparts in cultures. As illustrated in Fig. 7A
, the growth of prostatic stromal cells in coculture with
8b-LNCaP cells increased significantly compared with that of stromal
cells when grown with vector-LNCaP cells. The increase was
approximately 2-fold after 14 days of culture. The proliferation rate
of stromal cells in coculture with vector-LNCaP cells was, however,
similar to that of stromal cells grown alone. The growth rate of
8b-LNCaP cells in coculture was also higher than that of vector cells
cocultured with stromal cells. However, compared with the results with
LNCaP cells cultured alone (Fig. 3A)
, both 8b- and
vector-LNCaP cells showed inhibited growth in coculture with stromal
cells (Fig. 7B)
. To investigate whether the stimulatory
effect of stroma on 8b-LNCaP cells was prostate-specific, primary
stromal cells from human SV were also grown with the transduced LNCaP
cells. The cell morphology in coculture was similar to that of
prostatic stromal cell and LNCaP cocultures. After 10 days of culture,
the growth rate of these non-prostatic stromal cells in coculture with
8b-LNCaP cells increased 3-fold compared with that in coculture with
vector-cells (Fig. 7C)
. Again, there was no significant
difference in growth rate between SV stromal cells when cultured with
vector-LNCaP or cultured alone. The growth rate of 8b-LNCaP cells in
coculture with SV stroma was slightly higher than that of vector-cells
in coculture (Fig. 7D)
, but both cell types showed
remarkably restrained growth in the presence of SV stromal cells. It
was also noted that 8b-LNCaP cells and non-prostatic stromal cells
developed local confluency faster than those in coculture with
prostatic stromal cells and 8b-LNCaP cells. It was quite unique that SV
stromal cells became detached from the bottom of the culture flask once
local confluence was reached. This observation was confirmed by repeat
experiments with the same primary SV stromal cells.
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, the growth rate of stromal cells treated with CM from 8b-LNCaP cells
was clearly elevated compared with the use of vector-LNCaP cell CM
(P = 0.009).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Second, besides its ability to stimulate proliferation, it is also shown here that FGF8b can influence the various biological properties of the affected LNCaP cells. For example, in vitro parameters such as soft agar clonogenicity and matrigel invasion activity are significantly increased in 8b-LNCaP cells relative to the vector-LNCaP cells. An argument can be made that the observed changes of in vitro motility and invasion may be related partly to increased proliferation. However, this is unlikely to be a primary cause because the analyses were carried out only after 24 h, when proliferation is deemed to be quite limited. MMPs, which degrade extracellular matrix proteins, are known to be over-expressed in many types of cancers (33, 34, 35, 36, 37) . There are several reports that describe induction of MMP expression by FGF proteins in cancer cells, including prostate cancer cells, but not in normal epithelial cells (31 , 38, 39, 40, 41) . Thus it is possible that the switch between expression patterns of FGFR isoforms that occurs in prostate cancer cells, and that may be critical for abnormal proliferation by FGF8b, may also be responsible for activation of MMPs, thereby facilitating tumor cell invasion. The pleiotropic effect of FGF8b signaling is clearly documented here in the study of the in vivo tumorigenesis/diaphragm invasion assay. Although LNCaP cells were not tumorigenic in this assay, FGF8b expression converted them to be not only tumorigenic but also invasive in some animals during the 9-week period of observation. Taken together, evidence is presented that FGF8b overexpression confers tumorigenic and invasive properties to LNCaP cells. However, it remains to be demonstrated whether results of the study using a single cell line might have broader validity when other prostate cancer cells or prostate cancer tissues are evaluated.
Finally, when we examined the effect of FGF8b on the epithelial-stromal interactions in the setting of coculture, two important aspects were uncovered. It is clearly evident that the proliferation of the parental or transduced LNCaP cells is remarkably inhibited when cocultured with stromal cells from prostatic or non-prostatic tissues. In contrast, the growth of the stromal cells is strongly up-regulated in the presence of FGF8b-producing LNCaP cells but not in the presence of the control LNCaP cells. Although the autocrine regulation of LNCaP cells by FGF8b could compensate for the stromal effect to a degree, the negative effect of stroma still remains quite pronounced. The stimulatory effect of 8b-LNCaP cells on stromal cells seems not to be mediated by the released FGF8b because rFGF8b is not capable of stimulating stromal cells. Considering that other soluble molecules or factors induced and secreted from the FGF8b-stimulated LNCaP cells might be responsible, we used CM on stromal cells to examine the possibility. Because there is some positive effect of the CM on stroma, although far less than that observed in the context of coculture, this potential remains. Additional work will be necessary to characterize the released stimulatory factors to obtain a better definition of the role of FGF8b in the environment of the prostate. However, a stronger case could be made for the importance of cell-cell contact between epithelial and stromal cells for the observed stimulatory effect on the stroma. This contact effect is explicitly dependent on the presence of FGF8b-producing LNCaP cells. Thus, FGF8b signaling, beyond the release of soluble factors, appears to be critical in stromal proliferation. It is tempting to consider alterations of cell surface molecular expression in LNCaP cells from FGF8b-FGFR interactions as the primary inducer of stromal growth.
In summary, the effect of overexpression of FGF8b in LNCaP cells and their interaction with stromal cells may have broad implications in the progression of human prostate cancer. The autocrine stimulatory loop of FGF8b is likely to provide advantages to the neoplastic prostatic epithelium with respect to their proliferation and invasive properties. Additionally, it is speculated that, during the course of invasion and metastasis, the FGF8b producing malignant cells when in physical contact with stromal cells may stimulate growth of stromal cells. The accelerated proliferation of the stromal cells has, in fact, been suggested to provide an amenable milieu for the development and progression of cancer (42) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH R01 CA59705 and, in part, by a
grant from the T. J. Martell Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, University of Southern
California, Keck School of Medicine, 2011 Zonal Avenue, Los Angeles, CA
90089. Phone: (323) 442-1184; Fax: (323) 442-3049; E-mail: royburma{at}usc.edu
3 The abbreviations used are: FGF, fibroblast
growth factor; FGF8b, fibroblast growth factor 8, isoform b; CM,
conditioned medium; rFGF8b, recombinant FGF8b; VSV, vesicular
stomatitis virus; IRES, internal ribosome entry site; env,
envelope gene; GFP, green fluorescent protein; FBS, fetal bovine serum;
FACS, fluorescence-activated cell sorting; SV, seminal vesicle; MMP,
matrix metalloprotease.
Received 7/13/00. Accepted 9/29/00.
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)
in the 5' untranslated region are indicated. The transducing vector
contained the human FGF8b gene and a linked
GFP marker gene driven by the CMV promoter. An
IRES sequence was placed between the genes. This
expression cassette was flanked by the HIV long terminal repeats
(LTR) and contained the 
