Apoptosis induced by Fas (CD95) ligation is frequently lost during tumor progression; however, there is no direct evidence to support an association of Fas loss-of-function with metastatic tumor behavior. To determine whether Fas loss-of-function is critical for acquisition of
the metastatic phenotype, we have compared the ability of Fas-sensitive K1735 murine melanomas to form spontaneous lung metastases in wild-type and Fas ligand-deficient mice. Fas-sensitive melanoma clones are highly tumorigenic but rarely metastatic in wild-type syngeneic
mice. However, in Fas ligand-deficient mice, both the incidence and number of metastases are
increased. These findings provide the first evidence that Fas-Fas ligand interactions can suppress metastasis and that tumor Fas loss-of-function may be causally linked to metastatic progression.
Key words:
 |
Introduction |
Apoptosis fundamentally regulates tissue homeostasis.
Impairment of this process can result in aberrant cellular accumulation and increased tumor incidence (1). Fas
(CD95) and Fas ligand (FasL)1 are complementary receptor-ligand proteins that induce apoptosis in many cell
types. Fas is constitutively expressed in many tissues, whereas FasL is largely restricted to the spleen, lung, large and small intestine, uterus, testis, and structures of the eye (2, 3). In the lymphoid population, Fas-induced apoptosis has been shown to preserve immunologic tolerance, limit
clonal expansion, and maintain immunologic privilege (4).
The potential involvement of Fas in the control of malignant disease has been recognized only recently (5). Relevant to the current studies, we (8) and others (9, 10) have
reported that Fas-mediated apoptosis can be lost during
malignant progression as a consequence of Fas downregulation or impaired Fas signaling. Metastasis is the ultimate
step in the process of tumor progression and the principal
cause of death in cancer patients (11). In this report, we
identify Fas loss-of-function as a parameter both necessary and sufficient for acquisition of the metastatic phenotype.
To produce a clinically significant metastasis, tumor cells
must undergo a step-by-step sequence of events including
tissue invasion at the site of the primary lesion, entrance
into blood and lymphatics, avoidance of host immune defenses, survival in the circulation, adhesion to the vascular
endothelium, extravasation, and growth at a distant site
(12). During the metastatic process, tumor cells are subjected to intense selection pressures such that the majority
die (11, 12). Because each step in the metastatic cascade is
potentially rate limiting, failure to complete a single step
will render a tumor cell nonmetastatic. To determine potential restriction points in the metastatic process, we have
previously investigated the K1735 melanoma system comprised of a parental tumor with low metastatic capacity (13)
and clonally derived variants displaying enhanced or reduced metastatic properties compared to the parental tumor (14, 15). Using this tumor model, we have shown that
viable, genetically tagged nonmetastatic K1735 tumor cells
can disseminate from the subcutaneous site, survive transit
in the circulation and lymphatics, and extravasate into the
lung microenvironment (16). However, once in the lung, the nonmetastatic K1735 tumor cells fail to survive and
proliferate, with the subsequent failure to form metastatic
foci (16). An expansive body of literature has correlated
successful metastatic growth with tumor responsiveness to
paracrine and endocrine growth factors (11, 17, 18). However, in light of more recent evidence implicating apoptosis
in the control of tumor behavior (1, 5), we were led to
consider the potential role of tumor responsiveness to apoptosis-inducing factors in the microenvironment. Three
lines of circumstantial evidence led us to query whether
FasL regulated K1735 metastatic outgrowth in the lung.
First, FasL is present in the lung (2) and has been shown to
induce apoptosis in activated, Fas+ lymphocytes in vivo in
this organ microenvironment (19); second, nonmetastatic
and metastatic K1735 variants differ significantly in their
survival rate in lung conditioned media in vitro (20); and
third, the rate limiting step for K1735 metastasis has been documented to be outgrowth in the lung (16). Taken together, these results point toward a potential role for lung-derived FasL in the control of K1735 tumor growth in the
lung by a process involving Fas-mediated apoptosis.
 |
Materials and Methods |
Fas Expression on Murine Melanoma Clones.
Hamster anti-
murine Fas (Jo2) was purchased from PharMingen (San Diego,
CA). PE-conjugated goat anti-hamster antibody was purchased
from Caltag Labs. (Burlingame, CA). Indirect staining was carried
out as previously described (21) using 106 cells and 0.5 µg anti-Fas or isotype-matched control and a 1:50 dilution secondary antibody. Fas staining was determined on cells that had been fixed
overnight in 1% paraformaldehyde in PBS using a FACScan®
(Becton Dickinson, Mountain View, CA) with the windows set
to exclude dead cells and debris. 10,000 cells were examined for each determination.
Fas Sensitivity of Murine Melanoma Clones.
Fas sensitivity was
determined using the CD4+ T hybridoma line, 3A9 (22). 3A9
cells were activated by exposure to 10 ng/ml PMA (Sigma
Chemical Co., St. Louis, MO) and 3 µg/ml ionomycin (Sigma
Chemical Co.) for 4 h to upregulate FasL. After activation, 3A9
cells were washed three times and plated in quadruplicate in 96-well microtiter plates with 51Cr-labeled K1735 targets (5 × 103
cells/well) at a 50:1 effector/target ratio in a final volume of 200 µl calcium-free medium. Target cells were 51Cr labeled as previously reported (23). After 16-18 h incubation at 37°C in a 5%
CO2 atmosphere, 100-µl aliquots of cell-free supernatant were
harvested and radioactivity was measured using an LKB gamma counter (Amersham Pharmacia Biotech, Piscataway, NJ). Maximum lysis was determined by incubating 51Cr-labeled targets in 2 N
HCL. Percentage specific target cell lysis was calculated as follows:
100 × [(experimental lysis
spontaneous lysis)/(maximum lysis
spontaneous lysis)]. Lytic activity of activated 3A9 cells was
blocked by the inclusion of Fas-Fc (data not shown); unactivated
3A9 cells demonstrated no lytic activity. Anti-Fas killing was carried out by plating tumor cells at 5 × 103/well in triplicate and
adding 100 ng/ml biotinylated anti-Fas (Jo-2 clone; PharMingen)
or biotinylated isotype-matched control antibody (PharMingen)
for 30 min at 37°C then adding 200 ng/ml streptavidin (Jackson
ImmunoResearch Labs., West Grove, PA). After a 48-h culture
cell viability was determined using a tetrazolium reduction (MTT)
assay as described previously (8). Values shown represent the percentage of specific cytotoxicity in anti-Fas treated wells after subtracting that of the biotinylated, isotype-matched controls [(1
mean anti-Fas)/(mean isotype-matched control)] × 100.
Bioassay for FasL in Lung-conditioned Medium and FasL Detection
by Western Blotting.
Lung conditioned medium (LCM) was prepared according to the method of Szaniaswska et al. (24) with
modifications. In brief, murine lungs were surgically excised,
washed in cold PBS, cut into 1-2 mm3 pieces, washed in cold
HBSS, and suspended in serum-free MEM with 1% BSA and rotated at 37°C for 6 h. Initial medium was discarded and replaced
with MEM with 0.01% BSA, and the lung fragments were rotated at 37°C for an additional 24 h. After the final rotation, lung
fragments were removed and discarded and the remaining medium was spun at 15,000 g for 15 min. The cell-free supernatants were collected (LCM) and their protein concentration was determined. For the determination of bioactivity, K1735 cell lines and clones were plated in media supplemented with 0.3% fetal bovine serum and 65 µg/ml LCM at 106 cells/ml and cultured at 37°C, 5%
CO2 for 48 h. After culture, relative cellular viability was determined using the tetrazolium reduction (MTT) assay as previously
described by our laboratory (21). Background cell death was determined by culturing cells in media supplemented with 0.3% fetal bovine serum alone. Specificity of LCM killing was shown by
inclusion of either Fas-Fc (25) or isotype control-Fc (LT
R-Fc)
at 200 ng/ml for the 48-h culture period. The presence of FasL
protein in LCM was verified by Western blotting. For this procedure, 10-µg aliquots of LCM were separated by 12.5% SDS-PAGE gel and transferred to nitrocellulose membrane. FasL was
visualized by hybridization with a monoclonal anti-FasL (clone
33; Transduction Laboratories, Lexington, KY) and anti-mouse horseradish peroxidase was detected using the ECL system (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).
Detection of FasL in Murine Lung Sections by Immunohistochemistry.
Frozen sections of murine lung were fixed with 2% formaldehyde in PBS for 10 min and washed with PBS. Endogenous
peroxidase was blocked with 3% H2O2 in methanol for 12 min
and the sections were incubated with blocking solution (PBS supplemented with 5% horse serum and 1% goat serum) for 20 min.
Sections were then incubated with FasL-specific antibody, N-20
(Santa Cruz Biotechnologies, Santa Cruz, CA), at a 1:800 dilution in blocking solution overnight at 4°C. After washing, antibody staining was visualized using peroxidase-conjugated goat
anti-rabbit IgG, F(ab')2 (Jackson ImmunoResearch Labs.) at 1:500 in
blocking solution and stable diaminobenzidine (Research Genetics, Inc., Huntsville, AL). All sections were counterstained with
hematoxylin. Control sections were exposed to blocking solution alone.
Tumor Growth and Metastasis In Vivo.
C3H/gld and C3H/wt
mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). Single cell suspensions of K1735 melanoma cells were prepared by brief incubation of monolayer cultures in 0.02% EDTA
and 0.25% trypsin. Before injection, cells were resuspended in
PBS. For the determination of experimental metastases, 105 cells
were injected intravenously into the lateral tail vein and metastatic lung tumor growth was enumerated by visual inspection using light microscopy 3-5 wk after injection. For the determination of spontaneous metastases, mice were injected with 5 × 105
cells subcutaneously in the flank. Tumors were aseptically removed when their mean diameter reached 1.5 cm. Metastatic
lung growth was determined by visual inspection using light microscopy 4-6 wk later. Animal experimentation was performed in
accordance with institutional guidelines and the regulations and
standards of the USDA. Statistical analyses of metastatic incidence
in C3H and C3H/gld mice were carried out using a 2 × 2 contingency table of the number of mice with metastases versus the
number of mice without metastases using a one-sided
2 analysis.
Statistical analyses of the number of metastases in C3H versus
C3H/gld mice were carried out using a Mann-Whitney test.
 |
Results and Discussion |
If lung-derived FasL elicits the apoptosis of nonmetastatic K1735 tumors in vivo, then metastatic K1735 tumors
should lack Fas expression or demonstrate a resistance toward Fas-mediated apoptosis while their nonmetastatic
counterparts should retain Fas sensitivity. To test this
premise, we examined Fas expression and the ability to induce FasL-mediated cell death in vitro in the parental
K1735 tumor (K1735p) and five clonally derived tumor variants with well-defined metastatic properties. K1735p
(13) and the K1735 C-23 variant (14) rarely metastasize
from a subcutaneously growing tumor site and are considered to have low metastatic properties. In contrast, the
K1735 clones designated SW-1 and M-2 (15) are highly
metastatic to the lung, whereas their counterparts K1735 C-10 and C-19 are essentially nonmetastatic (14). As
shown in Fig. 1 A, Fas is highly expressed on both the low
and nonmetastatic K1735 tumors (K1735p, C-23, C-10
and C-19), whereas the highly metastatic clones SW-1 and
M-2 lack Fas expression. Although a striking correlation
existed between metastatic behavior and loss of Fas for the
highly metastatic K1735 clones, Fas was equivalently expressed on both the low and nonmetastatic K1735 tumors.
However, Fas expression does not necessarily predict cellular sensitivity to FasL-induced apoptosis (8, 9). To determine whether metastatic behavior in the low and nonmetastatic K1735 clones correlated with Fas sensitivity, tumor
targets were 51Cr-labeled and incubated with a FasL-expressing effector cell (22), and cytotoxicity was determined as
described in Materials and Methods. As shown in Fig. 1 B,
FasL was cytotoxic for all Fas-positive K1735 tumors. The
nonmetastatic clones C-19 and C-10 were most sensitive
to FasL-induced death, whereas the poorly metastatic tumors K1735p and C-23 were significantly more resistant to
such cytotoxicity. In three out of three experiments, the
low metastatic K1735 tumors were three- to fivefold more
resistant to FasL-induced cytotoxicity than were their metastatic counterparts. As expected, the highly metastatic M-2
and SW-1 clones lacking Fas expression were insensitive to
FasL-mediated killing. An identical sensitivity profile was
observed when anti-Fas-mediated killing was determined.
Representative clones are shown in Fig. 1 C. Therefore, Fas sensitivity rather than Fas expression appears to
uniquely differentiate among those tumors that are highly
metastatic, poorly metastatic, and nonmetastatic. The observation that Fas sensitivity and metastatic tumor behavior
are inversely correlated suggested a causal effect of Fas loss-of-function on tumor progression.

View larger version (26K):
[in this window]
[in a new window]

View larger version (21K):
[in this window]
[in a new window]

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
Fas expression and sensitivity of K1735 melanoma clones. (A) Flow cytometric analyses were carried out as described in Materials and Methods using a Fas-specific antibody (stippled line) and an isotype-matched control (solid line). Results represent two to five independent determinations for
each clone. (B) FasL-mediated lysis of K1735 clones in overnight 51Cr-release assay as described in Materials and Methods. Values represent mean ± SEM
of quadruplicate determinations within a single experiment. C10 and C19 killing were shown to be statistically different from that of K1735p and C-23
using Student's t test (C-10 versus K1735p, P = 0.017; C-10 versus K1735p, P = 0.003; C-10 versus C-23, P = 0.010; C-19 versus C-23, P = 0.016)
for the data shown. (C) Anti-Fas mediated killing of K1735 clones as described in Materials and Methods. Values represent mean ± SEM of triplicate determinations within a single experiment. Data are representative of two to five independent determinations for FasL and anti-Fas-mediated killing of
K1735 clones.
|
|
Although FasL mRNA is expressed in the lung (2), there
is scant knowledge about the distribution of FasL in this organ. To this end, we carried out immunohistochemical
staining of normal murine lung using a FasL-specific antibody. As shown in Fig. 2, A and B, the majority of FasL is
expressed on lung endothelial cells, with rare staining observed in type II pneumocytes as previously reported for
human lung tissue (26). To determine the bioactivity of
lung-expressed FasL, LCM was prepared as previously described (24). We have previously reported that LCM contains a cytotoxic factor that differentially induces cell death
in nonmetastatic K1735 tumors while sparing highly metastatic tumor variants (20). To ascertain whether such cytotoxicity was attributable to FasL, we examined LCM bioactivity against the nonmetastatic K1735 variant C-19 (14).
As shown in Fig. 3, potent, specific FasL-mediated cytotoxicity was observed in bioactive LCM preparations. Approximately 50% of control K1735 C-19 cells were nonviable after a 48-h culture in LCM. Fas-Fc, a competitive
inhibitor of FasL binding (25), almost completely inhibited
LCM-mediated killing of the K1735 C-19 cells. However,
in the presence of a non-FasL binding control such as the
lymphotoxin
receptor-Fc (LT
R-Fc), no such reversal of LCM cytotoxicity was observed. LCM-derived FasL was
additionally verified by immunoblotting. An intense band
of ~40 kD and an additional 32-kD mol mass form representing the nonglycosylated form of FasL (27) were consistently observed in bioactive LCM preparations (Fig. 2 C,
lane 3). Other immunoreactive lower molecular weight bands, possibly FasL degradation products, were observed
in some preparations (Fig. 2 C, lane 3). In nonbioactive
LCM preparations subjected to multiple freeze-thaw cycles, FasL was virtually undetectable by immunoblotting
(Fig. 2 C, lane 2), substantiating a direct relationship between LCM bioactivity and FasL protein.

View larger version (133K):
[in this window]
[in a new window]

View larger version (129K):
[in this window]
[in a new window]

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2.
FasL expression in the lung. Shown are staining of murine lung using isotype-matched control (A)
and FasL-specific antibody (B) visualized by peroxidase-labeled secondary antibodies as described in Materials
and Methods. (C) Western blot analysis of FasL protein in LCM. LCM was subjected to Western blotting using a FasL-specific antibody as described in Materials and Methods. Lane 1, control lysate from endothelial cells
(Santa Cruz Biotechnologies, Santa Cruz, CA); lane 2, LCM preparation subjected to multiple freeze-thaw cycles lacking FasL bioactivity; lane 3, LCM preparation used for the bioactivity assay shown in Fig. 3. The blot
shown is representative of four independent analyses of LCM.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Loss of K1735 C-19
viability after LCM culture is reversed by Fas-Fc. Cells were
plated in media supplemented
with 0.3% fetal bovine serum
and 65 µg/ml LCM. Fas-Fc or
isotype control-Fc (LT R-Fc)
were added at 200 ng/ml and
relative cellular viability was determined at 48 h using the tetrazolium reduction (MTT) assay.
Values shown were normalized
using values of cells cultured in media supplemented with 0.3% fetal bovine serum alone as background. Results are representative of two independent experiments.
|
|
If tumor Fas loss-of-function contributes to the development of the metastatic phenotype, then uncoupling Fas-
FasL interactions should permit the metastatic outgrowth
of Fas-sensitive tumors in the lung. To this end, we evaluated the metastatic behavior of the Fas-sensitive K1735 tumor and clones K1735p, C-23, and C-19 (Fig. 1) in normal (C3H) and FasL-deficient mice (C3H/gld). K1735
tumors were initially injected into the subcutis of both C3H or C3H/gld mice and regional growth was monitored. Although palpable tumors were usually detected earlier in C3H/gld mice than in C3H control mice, measurable tumor growth rates were nearly identical between the
two strains. Fig. 4 A shows one such growth curve using
the K1735p tumor. Furthermore, the mean tumor weights at the time of surgical resection 28 d after injection were
not statistically different between C3H and C3H/gld mice
for any of the tumors (Table 1). However, the incidence
and number of spontaneous lung metastases were increased
for all three tumors in mice lacking host-derived FasL. The
most striking enhancement of metastatic incidence and
number of metastases were observed using the K1735p tumor (Fig. 4 B, Table 1). This tumor showed macroscopic
lung metastases in 9 out of 10 C3H/gld mice (median 18, range 0-49), whereas only 3 out of 10 wild-type C3H
mice showed evidence of metastatic deposits (median 0, range 0-25). The increased incidence and number of metastases using the K1735p tumor was highly significant in
C3H/gld mice compared to C3H controls (difference in
incidence between C3H and C3H/gld P = 0.0003 using a
one-sided
2 test; difference in number of lung metastases
P < 0.0001 using a Mann-Whitney test). We next examined the metastatic growth of the clonally derived C-23
variant that, like K1735p, is considered to have low metastatic properties in C3H mice. As shown in Table 1, C-23
was metastatic in five out of seven C3H/gld mice (median 2, range 0-10), but only one out of five C3H mice (median
0, range 0-20), reflecting a significantly increased incidence
of metastasis in C3H/gld mice compared with wild-type
controls (difference in incidence between C3H and C3H/
gld mice P = 0.039, a one-sided
2 test). To address the effects of FasL loss-of-function on a nonmetastatic tumor, we
determined lung metastasis of the C-19 clone. As shown in
Table 1, the C-19 clone was spontaneously metastatic in
two out of five C3H/gld mice (range 0-16), whereas one
out of five C3H mice showed evidence of lung deposits
(range 0-1). Although not statistically significant with the
number of animals used for this experiment, these findings
again support a trend for increased incidence and number
of metastases in FasL-deficient animals. Moreover, these
data suggest that the loss of FasL is sufficient for acquisition
of the metastatic phenotype in a nonmetastatic tumor. A
potential criticism of such experiments is that the gld host
microenvironment is more permissive for metastatic tumor growth irrespective of the loss of FasL. If this were the case, then Fas-insensitive, metastatic tumors such as M-2 might
also demonstrate increased metastatic capacity in C3H/gld
animals. To test this premise, lung metastatic growth of M-2
was evaluated in C3H and C3H/gld mice. As shown in Table 1, lung metastases were not increased in FasL-deficient
mice compared with wild-type mice (difference in lung
metastases P = 0.421, Mann-Whitney test) documenting
that the gld lung environment per se is not inherently more
permissive for metastatic tumor outgrowth. Taken together, these results demonstrate that uncoupling of Fas-
FasL interactions can enhance tumor metastasis to organ
sites constitutively expressing FasL (K1735p, C-23) and
may be sufficient to confer metastatic phenotype in a nonmetastatic clone (C-19).

View larger version (65K):
[in this window]
[in a new window]

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Enhanced metastasis of K1735p melanoma cells in FasL-deficient (C3H/gld) mice. (A) 5 × 105 K1735p cells were injected
subcutaneously into the flank of C3H and C3H/gld mice. At indicated times, tumor diameters were determined by caliper measure.
Results shown represent mean ± SEM, n = 10, for each strain. (B)
Gross metastatic tumor growth in two representative lungs from
C3H/gld (left) and C3H control mice (right) subcutaneously injected
with K1735p cells.
|
|
A large body of evidence supports the concept that tumor cell growth and metastasis are dependent upon the interactions of the malignant cell and the host organ environment (17, 18). Our studies are commensurate with the
interpretation that the interactions of Fas and FasL on the
tumor and host environment, respectively, are critical determinants of tumor cell survival and growth in the metastatic lung site. Although the successful establishment of
metastatic lesions has been largely correlated with tumor
responsiveness to paracrine growth factors released in specific organ sites (17, 18), our data indicate an additional role
for apoptosis-inducing factors present in the specific microenvironment. As Fas-sensitive tumor cells would undergo apoptosis in organ sites constitutively expressing high
levels of FasL, we expect a priori that metastatic K1735 tumors preferentially colonizing the lung would be resistant
to Fas-mediated apoptosis. In this report, we document
that four out of four metastatic K1735 melanoma clones
show a loss of Fas expression and/or function compared with two nonmetastatic clones. We have observed a similar
loss of Fas expression and function in the highly metastatic
K1735 variants C-4 and C-2 (reference 14 and data not
shown). The highly metastatic lung-derived clones SW-1
and M-2 demonstrated a complete loss of both Fas expression and function, whereas the intermediate metastatic
clones K1735p and C-23 showed a significantly decreased
Fas sensitivity (Fig. 1). That Fas sensitivity was diminished but not entirely absent in K1735p and C-23 clones with
low metastatic capacity implies that even a partial loss of Fas
function may result in significant biologic consequences, as
has been reported for tumor development in lpr mice
where Fas expression is diminished (5). When Fas-FasL interactions are completely disrupted, such tumors may become significantly more metastatic (Table 1).
Initiation of Fas-mediated apoptosis is complex and
involves, at a minimum, levels of Fas sufficient to insure
optimal receptor cross-linking, the assembly of critical intracellular Fas-associated proteins necessary to initiate the
apoptotic cascade, and the presence or absence of "resistance" proteins such as bcl-xL, Fas-associated phosphatase 1 (FAP-1), and Flice inhibitory protein (FLIP) (9, 28). In
this respect, a balance between functional tumor-expressed Fas and host-derived FasL may be critical parameters regulating the capacity of a particular tumor to grow at a specified site. For example, Fas expression is absent on the
highly metastatic tumors K1735 SW-1 and M-2, precluding initiation of the apoptotic cascade by FasL cross-linking. Although Fas is highly expressed on low metastatic
clones K1735p and C-23, postreceptor alterations in Fas
signaling may prevent optimal initiation of the apoptotic cascade in response to FasL (Fig. 1, A and B). Similarly, the relative abundance of FasL in the microenvironment may
influence the capacity of a given tumor to survive or undergo apoptosis in disparate organ sites. In support of this
premise, constitutive FasL expression has been reported to
be considerably higher in the murine lung than in the skin
(3), providing a cogent explanation for the apparent conundrum that the Fas-sensitive K1735 tumors grew comparably in the subcutis of C3H and C3H/gld mice but were significantly more metastatic in the lung microenvironment. Although we favor the interpretation that an absence
of FasL in the lung microenvironment permitted the selective outgrowth of the Fas-sensitive K1735 tumors in C3H/
gld mice, it is conceptually possible that an absence of FasL-positive immune effector cells encountered in the blood
and lymphatics was also contributory (31). Although the latter possibility seems unlikely in view of the fact that viable, nonmetastatic tumor cells reach the lung microenvironment in normal mice (16), we are formally testing this
premise using bone marrow chimeras.
In summary, our studies provide the first evidence that
host-derived FasL can inhibit the malignant potential of
Fas-sensitive tumor cells and suppress organ-specific metastasis. The critical involvement of Fas-FasL in regulation of
the metastatic cascade further underscores the importance
of these proteins in homeostasis and prompts consideration
of strategies aimed toward modification of the tumor-host
environment to prevent metastatic outgrowth.
Address correspondence to Laurie Owen-Schaub, The Department of Immunology, University of Texas
M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 178, Houston, TX 77030. Phone: 713-792-2121; Fax: 713-745-1633; E-mail: lowensch{at}mdanderson.org
Supported in part by a National Institutes of Health (NIH) Cancer Center Support Core Grant 16672, an
institutional Physicians Referral Grant (to L.B. Owen-Schaub), a grant from the Skin Cancer Foundation at
the University of Texas M.D. Anderson Cancer Center (to L.B. Owen-Schaub), American Cancer Society
Grant CIM 88929 (to L.B. Owen-Schaub), and NIH fellowship F32 AI09351 (to L.L. Hill).
1.
|
Thompson, C.B..
1995.
Apoptosis in the pathogenesis and
treatment of disease.
Science.
267:
1456-1462
[Medline].
|
2.
|
Suda, T.,
T. Takahashi,
P. Golstein, and
S. Nagata.
1993.
Molecular cloning and expression of the fas ligand, a novel
member of the tumor necrosis factor family.
Cell.
75:
1169-1178
[Medline].
|
3.
|
French, L.E.,
M. Hahne,
I. Viard,
G. Radlgruber,
R. Zanome,
K. Becker,
C. Müller, and
J. Tschopp.
1996.
Fas and
Fas ligand in embryos and adult mice: ligand expression in
several immune-privileged tissues and coexpression in adult
tissues characterized by apoptotic cell turnover.
J. Cell Biol.
133:
335-343
[Abstract].
|
4.
|
Nagata, S..
1997.
Apoptosis by death factor.
Cell.
88:
355-365
[Medline].
|
5.
|
Peng, S.L.,
M.E. Robert,
A.C. Hayday, and
J. Craft.
1996.
A
tumor-suppressor function for Fas (CD95) revealed in T cell-
deficient mice.
J. Exp. Med.
184:
1149-1154
[Abstract].
|
6.
|
Zörnig, M.,
A. Grzeschiczek,
M.-B. Kowalski,
K.-U. Hartmann, and
T. Möröy.
1995.
Loss of Fas/APO-1 receptor accelerates lymphomagenesis in Eµ L-MYC transgenic mice
but not in animals infected with MoMuLV.
Oncogene.
10:
2397-2401
[Medline].
|
7.
|
Takeuchi, T.,
Y. Sasaki,
T. Ueki,
T. Kaziwara,
N. Moriyama,
K. Kawabe, and
T. Kakizoe.
1996.
Modulation of
growth and apoptosis response in PC-3 and LNCAP prostate-cancer cell lines by Fas.
Int. J. Cancer.
67:
709-714
[Medline].
|
8.
|
Owen-Schaub, L.B.,
R. Radinsky,
E. Kruzel,
K. Berry, and
S. Yonehara.
1994.
Anti-Fas on nonhematopoietic tumors:
levels of Fas/APO-1 and bcl-2 are not predictive of biological responsiveness.
Cancer Res.
54:
1580-1586
[Abstract].
|
9.
|
Irmler, M.,
M. Thome,
M. Hahne,
P. Schneider,
K. Hofmann,
V. Steiner,
J.-L. Bodmer,
M. Schröter,
K. Burns,
C. Mattmann, et al
.
1997.
Inhibition of death receptor signals by
cellular FLIP.
Nature.
388:
190-195
[Medline].
|
10.
|
Möller, P.,
K. Koretz,
F. Leithhäuser,
S. Brüderlein,
C. Henne,
A. Quentmeir, and
P.H. Krammer.
1994.
Expression
of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium.
Int. J. Cancer.
57:
371-377
[Medline].
|
11.
|
Fidler, I.J..
1990.
Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial
award lecture.
Cancer Res.
50:
6130-6138
[Abstract].
|
12.
|
Price, J.E.,
S.L. Aukerman, and
I.J. Fidler.
1986.
Evidence
that the process of murine melanoma metastasis is sequential
and selective and contains stochastic elements.
Cancer Res.
46:
5172-5178
[Abstract].
|
13.
|
Kripke, M.L..
1979.
Speculations on the role of ultraviolet radiation in the development of malignant melanoma.
J. Natl.
Cancer Inst.
81:
541-548
.
|
14.
|
Fidler, I.J.,
E. Gruys,
M.A. Cifone,
Z. Barnes, and
C. Bucana.
1981.
Demonstration of multiple phenotypic diversity
in a murine melanoma of recent origin.
J. Natl. Cancer Inst.
67:
947-956
[Medline].
|
15.
|
Talmadge, J.E., and
I.J. Fidler.
1982.
Enhanced metastatic
potential of tumor cells harvested from the spontaneous metastasis of heterogenous murine tumors.
J. Natl. Cancer Inst.
69:
975-980
[Medline].
|
16.
|
van Golen, K.L.,
S. Risin,
A. Staroselsky,
D. Berger,
M.A. Tainsky,
S. Pathak, and
J.E. Price.
1996.
Predominance of
the metastatic phenotype in hybrids formed by fusion of
mouse and human melanoma clones.
Clin. Exp. Metastasis.
14:
95-106
[Medline].
|
17.
|
Fidler, I.J..
1995.
Modulation of the organ environment for
treatment of cancer metastasis.
J. Natl. Cancer Inst.
87:
1588-1592
[Medline].
|
18.
|
Rusciano, D., and
M.M. Burger.
1992.
Why do cancer cells
metastasize to particular organs?
Bioessays.
14:
185-194
[Medline].
|
19.
|
Milik, A.M.,
V.A. Buechner-Maxwell,
J. Sonstein,
S. Kim,
G.D. Seitzman,
T.F. Beals, and
J.L. Curtis.
1997.
Lung lymphocyte elimination by apoptosis in the murine response to
intratracheal particulate antigen.
J. Clin. Invest.
99:
1082-1091
[Abstract/Free Full Text].
|
20.
|
van Golen, K.L.,
L.B. Owen-Schaub, and
J.E. Price.
1996.
Metastatic and nonmetastatic K1735 melanoma cells differ in
Fas-mediated apoptosis and growth response to lung-conditioned medium.
Proc. Am. Assoc. Cancer Res.
37:
A551
. (Abstr.)
.
|
21.
|
Owen-Schaub, L.B.,
S. Meterissian, and
R.J. Ford.
1993.
Fas/APO-1 expression and function on malignant cells of hematologic and nonhematologic origin.
J. Immunother.
14:
234-241
[Medline].
|
22.
|
Allen, P.M., and
E.R. Unanue.
1984.
Differential requirement for antigen processing by macrophages for lysozyme-specific T-cell hybridomas.
J. Immunol.
132:
1077-1079
[Free Full Text].
|
23.
|
Hill, L.L.,
B. Perussia,
P.A. McCue, and
R. Korngold.
1994.
Effect of human natural killer cells on the metastatic growth
of human melanoma xenografts in mice with severe combined immunodeficiency.
Cancer Res.
54:
763-770
[Abstract].
|
24.
|
Szaniawska, B.,
S. Majewski,
M.J. Kaminski,
K. Noremberg,
M. Swierz, and
P. Janik.
1985.
Stimulatory and inhibitory activities of lung-conditioned medium on the growth of normal
and neoplastic cells in vitro.
J. Natl. Cancer Inst.
75:
303-306
[Medline].
|
25.
|
Crowe, P.D.,
T.L. Van Arsdale,
B.N. Walter,
K.M. Dahms, and
C.F. Ware.
1994.
Production of lymphotoxin (LT alpha)
and a soluble dimeric form of its receptor using the baculovirus expression system.
J. Immunol. Methods.
168:
79-89
[Medline].
|
26.
|
Niehans, G.A.,
T. Brunner,
S.P. Frizelle,
J.C. Liston,
C.T. Salerno,
D.J. Knapp,
D.R. Green, and
R.A. Kratze.
1997.
Human lung carcinomas express Fas ligand.
Cancer Res.
57:
1007-1012
[Abstract].
|
27.
|
Mariani, S.M.,
B. Matiba,
C. Bäumler, and
P.H. Krammer.
1995.
Regulation of cell surface APO-1/Fas (CD95) ligand
expression by metalloproteases.
Eur. J. Immunol.
25:
2303-2307
[Medline].
|
28.
|
Muzio, M.,
A.M. Chinnaiyan,
F.C. Kischkel,
K. O'Rourke,
A. Shevchenko,
J. Ni,
C. Scaffidi,
J.D. Bretz,
M. Zhang,
R. Gentz, et al
.
1996.
FLICE, a novel FADD-homologous ICE/
Ced-3-like protease, is recruited to the CD95 (Fas/APO-1)
death inducing signalling complex.
Cell.
85:
817-827
[Medline].
|
29.
|
Sato, T.,
S. Irie,
S. Kitada, and
J.C. Reed.
1995.
FAP-1: a
protein tyrosine phosphatase that associates with Fas.
Science.
268:
411-415
[Medline].
|
30.
|
Boise, L.H.,
A.J. Minn,
P.J. Noel,
C.H. June,
M.A. Accavitti,
T. Lindsten, and
C.B. Thompson.
1995.
CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL.
Immunity.
3:
87-98
[Medline].
|
31.
|
Arase, H.,
N. Arase, and
T. Saito.
1995.
Fas-mediated cytotoxicity by freshly isolated natural killer cells.
J. Exp. Med.
181:
1235-1238
[Abstract].
|
32.
|
Liles, W.C.,
P.A. Kiener,
J.A. Ledbetter,
A. Aruffo, and
S.J. Klebanoff.
1996.
Differential expression of Fas (CD95) and
Fas ligand on normal human phagocytes: implications for the
regulation of apoptosis in neutrophils.
J. Exp. Med.
184:
429-440
[Abstract].
|
33.
|
Keiner, P.A.,
P.M. Davis,
G.C. Starling,
C. Mehlin,
S.J. Klebanoff,
J.A. Ledbetter, and
W.C. Liles.
1997.
Differential induction of apoptosis by Fas-Fas ligand interactions in human
monocytes and macrophages.
J. Exp. Med.
15:
1511-1516
.
|