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Tumor Biology |
Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland [F. C. B., C. B., A. F. G. Q.], and Chirurgische Universitätsklinik, D-39120 Magdeburg, Germany [M. A. R.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since its identification (5 , 6) , caveolin-1 has been implicated in the process of cell transformation: caveolin-1 is a major substrate for phosphorylation on tyrosine upon cell transformation by the Rous sarcoma virus (6) ; caveolin-1 mRNA and protein levels are reduced in NIH-3T3 fibroblasts transformed by several oncogenes (7) ; and caveolin-1 levels are reduced in a variety of carcinoma cell lines, including human mammary carcinoma (8) and lung carcinoma cells (9) . These results suggest that reduced caveolin-1 expression may represent a general characteristic or even a requirement of transformed cells and that caveolin-1 could play a central role as an inhibitor of tumor formation.
Caveolin-1 interacts directly with and inhibits or sequesters the
inactive form of many key signaling molecules including
heterotrimeric G proteins, Ha-Ras, c-Src, endothelial nitric oxide
synthase, protein kinase C 
,
MAPK,3
and tyrosine kinase receptors via a motif referred to as the
scaffolding domain (10, 11, 12, 13, 14, 15)
. Additionally, many of the
aforementioned proteins contain a consensus motif for caveolin-1
binding (16
, 17)
. Thus, caveolin-1 may reduce cell
tumorigenicity by virtue of its ability to bind to and inhibit or
sequester inactive forms of signaling proteins including oncogenes.
Consistent with this hypothesis, caveolin-1 levels are reduced in
oncogenically transformed cells (7)
, reexpression of
caveolin-1 abrogates anchorage-independent growth in
oncogene-transformed cells (18)
, and down-regulation of
caveolin-1 by overexpression of caveolin-1 antisense RNA is sufficient
to transform NIH-3T3 cells (19)
.
Despite such evidence favoring a role for caveolin-1 as a tumor suppressor gene in several cell systems, no mutations were found in human cancer cells. In addition, CpG islands associated with the caveolin-1 gene are not methylated in either primary tumors or tumor-derived cell lines in which caveolin-1 expression is low (20) . Thus, although reduced caveolin-1 expression can clearly be linked to increased tumorigenicity in some cell systems, classification of caveolin-1 as a tumor suppressor remains controversial at this point.
Only a few studies have directly investigated caveolin-1 changes on tumor formation in humans and how reexpression of caveolin-1 modulates the tumor-forming ability of tumor-derived cells. In this study, these questions were addressed with a focus on the role of caveolin-1 in human colon cancer. The results presented show that: (a) caveolin-1 mRNA and protein levels were reduced in human colon carcinoma cell lines, as well as in human colon tumors; (b) reexpression of caveolin-1 in the colon carcinoma cell lines HT29 and DLD1 significantly reduced the tumorigenicity of these cells when they were injected in nude mice; and (c) tumor formation in vivo resulted in the selection of cells with lower basal caveolin-1 levels. To our knowledge, this is the first study showing that caveolin-1 reexpression in human carcinoma cells reduces their ability to form tumors in nude mice.
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MATERIALS AND METHODS |
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Cell Culture.
The human colon carcinoma cell lines SW480, SW620, Co112, HT29 and its
derived differentiated clones HT29-5 M12 and HT29-5 M21
(21)
, LoVo and the LoVo clones E2 and C5 (selected for
higher metastatic potential; Ref. 22
) were provided by Dr.
Bernard Sordat (ISREC, Epalinges, Switzerland). Cell line DLD1 was
provided by Dr. Emanuela Felley-Bosco (Institute of Pharmacology,
University of Lausanne, Lausanne, Switzerland), and the cell lines
Caco2 and MDCK strain II were provided by Dr. Walter Hunziker
(Institute of Biochemistry, University of Lausanne, Lausanne,
Switzerland). NIH-3T3 fibroblasts and NIH-3T3 ExTu, a population of
NIH-3T3 cells isolated after tumor formation on nude mice
(23)
, were provided by Dr Ernst Reichmann (ISREC). HT29,
HT29-5 M12, HT29-5 M21, Co112, Caco2, MDCK, NIH-3T3, and NIH-3T3 ExTu
cells were cultured in DMEM supplemented with 10% FCS and penicillin,
streptomycin, and neomycin. LoVo and LoVo clones were cultured in the
same medium containing 0.1%
Na2CO3. SW480, SW620, and
DLD1 cells were maintained in RPMI 1640 with 10% FCS and antibiotics
as described above. All cells were cultured at 37°C under 5%
CO2 and passaged every week using trypsin/EDTA.
Isolation of Human Colon Crypts and Purification of Epithelial
Cells.
Human colonic crypts and, subsequently, colonic epithelial cells or
stroma were isolated as described previously (24
, 25)
after obtaining the informed consent of the patients. Operations were
performed at the University Hospital of Geneva (Geneva, Switzerland)
and at the Carl-Thiem Klinikum (Cottbus, Germany). Authorization
was provided by the Ethics Committee. Epithelial cell viability after
purification was >90% as determined by trypan blue staining. After
cross-staining with a pan-anticytokeratin antibody (CAM 5,12;
Perkin-Elmer, Norwalk, CT), epithelial cell preparations were
shown to be >95% pure by fluorescence-activated cell-sorting
analysis.
Plasmids.
Plasmid placIOP-cav-1, which allows IPTG-inducible expression of
caveolin-1 in transfected cells, was constructed as follows. The
full-length cDNA encoding dog caveolin-1 was amplified by reverse
transcription-PCR using caveolin-1-specific primers flanked by
NotI restriction sites and RNA isolated from MDCK cells as a
template. The resulting cDNA was purified and then cloned into the
NotI site of placIOP, which consists of vectors p3'SS and
pOPRSVI chloramphenicol acetyltransferase from Invitrogen
(Carlsbad, ) fused together as described previously
(23)
. The sequence of the 5' sense primer, which also
included a Kozak motif (underlined) upstream of the
initiation ATG codon, was
5'-CCGAGCGCGGCCGCCATGTCTGGGGCAAATAC-3', and the
sequence of the antisense primer was
5'-TATCTGGCGGCCGCTTATGTTTCTTTC-TGCATGTTG-3'. NotI
restriction sites are indicated in bold. The construct
pGEM-cav-1 was used to produced caveolin-1-specific probes for Northern
analysis and was obtained by amplifying a cDNA sequence conserved
between dog and human (nucleotides 63–433 of the cDNA coding sequence)
by reverse transcription-PCR using RNA isolated from MDCK cells as a
template and appropriate primers to allow subsequent cloning of the
amplified product into the XbaI/EcoRI sites of
pGEM2 (Promega, Madison, WI). The sense primer
(5'-GGGCAACATCTAGAAGCCCAACAAC-3') included a XbaI
site (shown in bold). The antisense primer
(5'-CTGATGCACTGAATTCCAATCAGGAA-3') contained an
EcoRI site (shown in bold). The pSP65m-ß-actin
plasmid (26)
used for standardization of Northern blots
was kindly provided by Markus Nabholz (ISREC).
Stable Transfection of HT29 and DLD1 Cells with a Plasmid
Permitting Inducible Expression of Caveolin-1.
HT29 and DLD1 cells were stably transfected with placIOP (mock) or
placIOP-cav-1 by calcium phosphate precipitation as described
previously (27)
. Individual clones resistant to 500
µg/ml hygromycin B were screened for IPTG-induced expression of
recombinant caveolin-1 by Western blot analysis. Induction of
caveolin-1 was maximal after 24 h of stimulation with 1
mM IPTG.
Northern Analysis.
Total cellular RNA was extracted with a purification kit in the
presence of guanidinium thiocyanate (Qiagen, Hilden, Germany) according
to the manufacturer’s instructions. Samples containing 15 µg of
cytoplasmic RNA were fractionated on 1% agarose gels prepared in 10
mM sodium phosphate buffer (pH 7), transferred to a nylon
membrane, and cross-linked to the membrane by UV irradiation as
described previously (28
, 29) . Alternatively, multiple
tissue or cell line Northern blots were purchased from Clontech
Laboratories (Palo Alto, CA). After overnight preincubation at 55°C
in hybridization buffer [50% formamide, 5x SSC, 1 mM
EDTA, 0.2% SDS, 2x Denhardt’s solution, 0.5 mg/ml yeast tRNA, and
0.25 mg/ml salmon sperm DNA in 50 mM sodium phosphate
buffer (pH 6.5)], blots were further incubated for 24 h with
106 cpm/ml 32P-labeled RNA
probes for caveolin-1 in hybridization buffer. Probes were synthesized
as described previously (28
, 29)
from
XbaI-linearized pGEM-cav-1. Blots were washed four times
(for 15 min each time) at 65°C in 0.1x SSC/0.1% SDS solution
and exposed to film (BioMax MR-1; Kodak, Rochester, NY). After
caveolin-1 detection, blots were stripped (according to a protocol from
Clontech Laboratories) and standardized to ß-actin using a ribo-probe
prepared from MaeI-linearized pSP65m-ß-actin.
SDS-PAGE and Western Blotting.
Expression of caveolin-1 in carcinoma cell lines, transfected HT29 or
DLD1 cells, human colon tissues, or NIH-3T3 cells was studied by
Western blot analysis. Cells were grown until they reached 80%
confluence. Culture medium was then removed, and the cells were washed
twice with cold PBS and lysed in buffer containing 4% SDS, 125
mM Tris-HCl (pH 6.8), and protease inhibitors (10 µg/ml
benzamidine, 2 µg/ml antipain, and 1 µg/ml leupeptin). Cell lysates
were sonicated, and the protein concentration was determined with the
BCA assay. Human colon tissues were lysed in a similar fashion, but
homogenates were passed through a 25-gauge needle several times,
sonicated, and cleared by centrifugation for 5 min at 10,000 x g in an Eppendorf centrifuge. The protein
concentration of supernatants was determined by the BCA assay. All
samples were adjusted to Laemmli buffer composition [Ref.
30
; 2% SDS, 10% glycerol, 62.5 mM
Tris-HCl (pH 6.8), 100 mM DTT, and 0.1%
bromphenol blue], denatured by heating at 95°C for 5 min, and
subsequently loaded on 10% gels. After separation, proteins were
transferred onto nitrocellulose. Membranes were stained with Ponceau
Red S (Sigma) to verify equal loading of samples and blocked overnight
in PBS/3% milk/2 mM NaN3.
The membranes were then incubated for 1 h at room temperature with
either anti-caveolin-1 (1:10,000) or anti-actin (1:1,000) antibodies
diluted in blocking solution. Membranes were then washed five times in
PBS/0.1% Tween 20, incubated for 1 h with a second antibody
(1:2,500) diluted in blocking solution (no azide), and washed again as
described previously. Membrane-bound second antibodies were detected by
enhanced chemiluminescence following the instructions of the
manufacturer.
Tumorigenicity Assays.
Cells (106) were suspended in 50 µl of DMEM and
injected s.c. into 6–8-week-old nude mice. For each mouse, control
cells (parental HT29 or DLD1 cells or mock-transfected cells) were
injected on the left, and HT29 or DLD1 cells transfected with
caveolin-1 [clones C13, C14, C16 (HT29 clones) or C2 or C4 (DLD1
clones)] were injected on the right. Large (D) and
small (d) diameters of growing tumors were measured twice a
week, and the corresponding volumes (V) were estimated using
the equation V = d2 x D x 
/6. To reisolate tumor cells for further culture, the tumor
tissue was excised, cut into small pieces under sterile conditions
using scalpel blades, and digested with trypsin/EDTA for 15 min at
37°C. Tumor cells were cultured until confluence in 10-cm Petri
dishes, trypsinized, diluted 1:10 in fresh medium, and seeded again.
After a second passage, when tissue debris and contaminating cells had
been eliminated, ex-tumor cells were lysed at 80% confluence and
processed for caveolin-1 detection as described previously.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, top panel) were compared by Northern blotting analysis. The
3-kb specific mRNA of caveolin-1 (22)
was extremely
abundant in the heart but undetectable in peripheral blood leukocytes,
which served in these experiments as positive and negative controls,
respectively (22
, 32)
. Interestingly, caveolin-1 mRNA
levels in the small intestine or colon were 10–20-fold higher than the
levels detected in the colon carcinoma cell line SW480.
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. Caveolin-1 protein levels (Fig. 2A)
were extremely low in
all cell lines analyzed, as compared with the levels in MDCK cells, and
never exceeded the levels observed in SW480 cells. Expression
was particularly low in HT29, Co112, and Caco2 cells. Even in SW480,
SW620, and DLD1 cells, in which caveolin-1 was present, levels were
50–100-fold lower than that in MDCK cells. In addition, all colon
carcinoma cell lines had low levels of caveolin-1 mRNA (Fig. 2B)
that were comparable to or lower than those observed in
SW480 cells (Fig. 1)
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, whereby colon mucosa (epithelium) and stroma from either normal or
tumor tissue were compared. Caveolin-1 levels were decreased in tumor
mucosa, suggesting that low levels of caveolin-1 expression detected in
colon carcinoma lines reflect an inherent property of the immortalized
cells derived from colon tumor epithelium. In addition, caveolin-1
levels were similarly decreased in tumor stroma (see "Discussion").
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are equivalent to the ratio of scanning densitometry values obtained
for normal versus tumor tissue. In about 70% (10 of 15) of
the samples analyzed, caveolin-1 levels were reduced up to 7-fold
(average, 3.9-fold) in mucosa. For stroma, fewer samples were
available, but, on average, a similar decrease in caveolin-1 presence
was observed. In about 30% (5 of 15) of the cases, no significant
decrease of caveolin-1 expression was observed in tumor tissue, and the
trend was even reversed in one situation. Taken together, these results
suggest that caveolin-1 expression levels are reduced in human colon
tumor samples as well as in colon carcinoma cell lines and that the
decreased presence of the caveolin-1 protein may be attributed to
reduced mRNA levels.
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and mRNA (Fig. 4B)
. Thus, tumor formation in
mice correlated with either reduction of caveolin-1 expression in
NIH-3T3 cells or elimination of cells expressing caveolin-1.
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. The clones C13, C14, and C16 expressed higher caveolin-1 levels than
parental HT29 or mock-transfected cells, even when grown in the absence
of IPTG (Fig. 5A
, -IPTG). The
addition of IPTG (Fig. 5A
, +IPTG) dramatically increased the
basal level of caveolin-1 expression in all clones but had no effect on
both parental and mock-transfected HT29 cells. Basal caveolin-1
expression levels and levels after IPTG induction were not identical in
these clones, with clone C14 expressing the highest amount, and clone
C16 expressing the lowest amount. Similarly, clones C2 and C4 expressed
higher caveolin-1 levels than parental DLD1 and mock-transfected cells,
but caveolin-1 expression levels were not increased by the addition of
IPTG (Fig. 5B)
.
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and 7
). All HT29 cells led to noticeable tumor formation 1 month after
injection, but in 75% (n = 10) of the cases
studied, tumors were either significantly smaller for
caveolin-1-expressing HT29 clones (Fig. 6, A and B
; n = 7) or almost undetectable
(Fig. 6C
; n = 3). Where tumors
developed, the kinetics of tumor formation were different, with a lag
time of 2–3 weeks before tumor formation was noticeable (Fig. 6, A and B)
. For 25% (n = 3) of the mice tested, however, no difference was detected in either
the size of the tumors or the kinetics of tumor development (Fig. 6D)
.
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, A–C). As for HT29-cav-1
clones, tumor formation was generally observed after an initial lag
period of 2–3 weeks (Fig. 7, B and C)
. For 30%
of the mice tested (n = 2), however, no
difference in the size of the tumor was detected (Fig. 7D)
.
Caveolin-1 Expression Levels in HT29-cav-1 and DLD1-cav-1 Cells
Were Reduced on Tumor Formation in Nude Mice.
The experiments with NIH-3T3 fibroblasts (Fig. 4)
revealed that
tumor formation resulted in cell populations with reduced caveolin-1
levels. Thus, the possible explanations for why tumor formation had
occurred in some cases with transfected HT29 and DLD1 cells were that
this process may have led to either elimination of caveolin-1
expression, despite being under the control of an exogenous promoter,
or to selection of cells with lower basal levels of caveolin-1
expression. To investigate these possibilities, cells were isolated
from excised tumors and put back in culture, and, after pure cell
populations were available, cells were examined for caveolin-1 protein
expression (Fig. 8
, ExTumor). Directly after plating, cells derived from tumors
were a mixture of host cells (mainly fibroblasts) and tumor cells, but
only tumor cells underwent rapid proliferation. By contrast, host cells
tended to detach and die rapidly (data not shown). When culture plates
were confluent after two passages, homogenous tumor cell populations
that were morphologically identical to parental HT29 or DLD1 cells but
had the additional ability to grow in the presence of hygromycin B were
obtained (data not shown). In these cells, basal levels of caveolin-1
expression were reduced when compared with those observed for
HT29-cav-1 cells before injection into mice (Fig. 8
, ExTumor
and BI, respectively). Nevertheless, caveolin-1 expression
could still be induced by the addition of IPTG (data not shown). Thus,
selection for HT29 cells expressing lower levels of caveolin-1 occurred
on tumor formation in nude mice. Similar results were obtained with
DLD1 cells (data not shown).
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. However, caveolin-1 was not detectable in Caco2 cells
cultured normally with frequent passaging (proliferative,
undifferentiated cells; see Fig. 2
) or in cells left for 5 weeks
without passaging (differentiated cells; data not shown; Ref.
34
). Taken together, these experiments suggest that
variations in culture conditions favoring differentiation have little
effect on caveolin-1 expression in colon carcinoma cells but that
phenotypic changes correlate with increased caveolin-1 expression when
observed in conjunction with drug resistance.
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, suggesting that up-regulation of caveolin-1
might occur during metastasis. |
DISCUSSION |
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In complete agreement with this concept, caveolin-1 was identified as 1 of 26 candidate tumor suppressor genes in human mammary carcinomas using differential display and subtractive techniques (31) . In addition, the caveolin-1 gene has been mapped to a tumor suppressor locus in both the human (locus D7S522) and mouse (locus 6-A2/731) genomes (36 , 37) . These regions are frequently deleted or contain breakpoint sites for chromosome translocation in a wide variety of tumors (36 , 38) . Furthermore, caveolin-1 was recently identified as a target protein for p53-dependent regulation (39) . However, at the DNA level, there is virtually no evidence that caveolin-1 is a tumor suppressor gene because the caveolin-1 gene is neither mutated nor methylated in cancer cells (20) , although methylation of the caveolin-1 promoter has been described in breast cancer cell lines (37) .
Despite this wealth of information, few studies directly investigated
how expression levels of caveolin-1 change on tumor formation in either
mice or humans. In this study, we focused in particular on the role
that caveolin-1 may play in human colon cancer. Our results showed that
caveolin-1 mRNA and protein levels (Figs. 1
and 2)
were reduced in
colon carcinoma cell lines as compared with normal colon tissue. Thus,
after lung (9)
and possibly breast (8)
carcinomas, colon carcinomas represent a third group of human
carcinomas in which caveolin-1 levels are reduced as a consequence of
what appears to be predominantly transcriptional regulation. Moreover,
the comparison of samples from normal colon and colon tumor tissue
revealed that caveolin-1 protein expression was reduced in tumor
epithelium, thereby establishing a direct link between the reduced
caveolin-1 expression levels observed in human colon carcinoma cell
lines and a reduction of caveolin-1 expression observed in colonic
epithelial cells on tumor formation.
Caveolin-1 down-regulation was not only observed in colon tumor mucosa but was also observed in the adjacent stroma, suggesting that carcinoma cells may be able to modulate expression levels of caveolin-1 in surrounding tissues, constituted predominantly of adipocytes and endothelial and muscle cells. In this context, it is interesting to note that angiogenesis activators such as vascular endothelial growth factor, basic fibroblast growth factor, and HGF down-regulate caveolin-1 in human endothelial cells (40) .
To corroborate the notion that tumor formation correlates with
reduction of caveolin-1 expression, caveolin-1 levels were compared in
the parental NIH-3T3 cells and after tumors had developed in nude mice.
Tumor formation in this experimental model system yielded a cell
population with less caveolin-1 (Fig. 4)
, indicating that caveolin-1
may be rate-limiting for anchorage-independent growth and tumor
formation in mice. Consistent with this idea, NIH-3T3 ex-tumor cells
with lower caveolin-1 levels formed tumors more rapidly on reinjection
into nude mice (data not shown).
Whereas our results are in perfect agreement with those obtained in mammary (8) and lung carcinoma cell lines (9) , they disagree with results obtained by others for prostate and breast cancer (41 , 42) . There, elevated expression of caveolin-1 was associated with development of prostate and breast cancers in both the human and mouse systems (41) . Alternatively, Hurlstone et al. (20) failed to detect any difference between the expression of caveolin-1 in tumor cells versus normal breast tissue. These results indicate that the role of caveolin-1 may vary considerably, depending on the tissue involved.
Interestingly, caveolin-1 levels were highest in metastases derived
from primary prostate tumors, suggesting that accumulation of
caveolin-1 relative to normal epithelium occurred with progression of
prostate cancer (41)
. One major difference in that respect
between prostate and colon tissue is that caveolin-1 mRNA and protein
are present at high levels in normal colon epithelium (Figs. 1
2
3)
,
whereas only minimal expression is observed in the corresponding
prostate tissue samples (41)
. Thus, transformation and
progression of malignancy in cells that normally express caveolin-1 may
occur in two phases: (a) initially, down-regulation of
caveolin-1 is required during primary tumor formation; and
(b) subsequently, up-regulation of caveolin-1 may occur in
methotrexate-resistant HT29 cells (Fig. 9A)
as well as in
multidrug-resistant human colon carcinoma HT29 cells and breast
carcinoma MCF-7 cells (43)
. Alternatively, reexpression of
caveolin-1 may be required during metastasis. In support of this idea,
colon carcinoma clones selected from the LoVo cell line for higher
metastatic potential (35)
had elevated caveolin-1 protein
levels when compared with parental cells (Fig. 9B)
.
Here, however, two additional points need to be considered. First,
basal caveolin-1 levels are already higher in LoVo than in other colon
carcinoma lines (see Fig. 9B
); second, although the cell
populations E2 and C5 were obtained by sequential injection into mice
followed by isolation of cells from resulting lung metastases, this
does not mean that metastases generally have higher levels of
caveolin-1 expression than the original tumor. For instance, the
primary colon tumor SW480 cells and matched metastatic colon cancer
SW620 cells originating from the same patient both have equally low
caveolin-1 levels (Fig. 2)
. Similarly low caveolin-1 levels were also
observed (data not shown) for liver (Isreco2) and peritoneal (Isreco3)
metastases derived from a primary ascending human colon cancer
(Isreco1), cell lines characterized by Cajot et al.
(44)
. Taken together, this would argue that control of
caveolin-1 levels in colon carcinomas is complex and that no simple
unifying hypothesis is currently available to explain all available
observations. Clearly, more research is required to address these
issues. In conclusion, down-regulation of caveolin-1 might be an early
event that occurs in primary tumor formation of a limited set of
epithelia that normally express high levels of caveolin-1, including
colon (this study) and lung (9)
.
The precise mechanism by which reduced levels of caveolin-1 expression
in epithelium would promote initial steps toward carcinoma formation is
not clear. Several reports indicate that caveolin-1 possesses a
specific motif, referred to as the scaffolding domain, that can bind to
and inhibit the activity of a number of proteins involved in signal
transduction, including heterotrimeric G proteins (11)
,
Src family tyrosine kinases (10)
, endothelial nitric oxide
synthase (45, 46, 47, 48)
, Neu tyrosine kinase (49)
,
epidermal growth factor receptor (50)
, and protein kinase
C (51)
. Thus, reduced levels of caveolin-1 would
prolong cell stimulation linked to one of these numerous signal
transduction pathways. Consistent with this notion, targeted
down-regulation of caveolin-1 in NIH-3T3 cells leads to hyperactivation
of the p42/p44 MAPK pathway and, as a consequence, cell
transformation (19)
. However, overexpression of caveolin-1
inhibited both MAPK-dependent and -independent pathways in adipose
cells, whereas in Cos-7 cells, caveolin-1 enhanced MAPK-dependent
signaling (52)
. Thus, modulation of the MAPK signaling
pathway as well as other signaling pathways by caveolin-1 may be
differentially regulated, depending the cell system studied. In
addition, caveolin-1 levels are likely to be tightly controlled in
cells because both up- and down-regulation alter cell signaling events.
More recently, caveolin-1 expression was reported to inhibit transcription of the cyclin D1 gene, suggesting that loss of caveolin-1 expression during tumorigenesis may lead to cellular transformation via the ß-catenin/TCF/LEF signaling pathway (53 , 54) . Caveolin-1 is also involved in signal transduction events mediated by several integrins on binding to extracellular matrix proteins. There, caveolin-1 plays a key role by linking integrins to Fyn activation, which in turn is responsible for Shc recruitment, regulation of Ras-MAPK signaling, and cell cycle progression (55) . Thus, anchorage-independent growth observed in transformed cells on down-regulation of caveolin-1 may be linked to this particular aspect of caveolin-1 function.
Finally, direct evidence for the importance of caveolin-1 in limiting
the tumor-forming ability of colon carcinoma cells is provided here
(Figs. 6
and 7)
. Expression of caveolin-1 in transfected HT29 and DLD1
clones generally reduced the size of tumors formed on injection into
nude mice and delayed the onset of tumor formation in most cases (Figs. 6
and 7)
. When tumors were detectable, their presence correlated with a
decrease in basal caveolin-1 expression in comparison with the levels
detected before cell injection into mice (Fig. 8)
. These observations
provide strong support for the notion that an initial period of
selection exists. Those cells that have lower caveolin-1 levels and/or
succeed in reducing caveolin-1 expression subsequently proliferate and
are able to form tumors in nude mice.
The reduction of caveolin-1 mRNA levels observed in breast and lung
tumor cell lines indicates that caveolin-1 down-regulation occurs
primarily at the transcriptional level. The caveolin-1 gene is
reportedly not methylated in either breast primary tumors or
tumor-derived cell lines, indicating that the observed down-regulation
of caveolin-1 mRNA expression in breast tumors does not result from
transcriptional silencing or DNA methylation during tumor progression
(20)
. However, those results are controversial because
Engelman et al. (37)
identified a CpG island
within the caveolin-1 promoter region that was methylated in human
breast cancer cell lines. Also, in our studies, reduction of the mRNA
levels appeared to be an important mechanism by which caveolin-1
protein levels were regulated because both were dramatically
reduced in colon carcinoma cell lines as compared with the levels
observed in normal colon tissue (Figs. 1
and 2)
.
In summary, the experimental data presented here show that: (a) caveolin-1 protein levels were reduced in colon tumors from human patients; (b) colon carcinoma cells had low levels of caveolin-1 mRNA and protein; (c) expression of caveolin-1 in the colon carcinoma cell lines HT29 and DLD1 blocked or retarded tumor formation in nude mice; (d) the ability of HT29-cav-1 and DLD1-cav-1 (and also NIH-3T3 cells) to form tumors in nude mice despite the initial presence of caveolin-1 was linked to a selection process favoring the proliferation of those cells with reduced basal caveolin-1 levels; and (e) initial caveolin-1 down-regulation in colon carcinoma cells need not be an entirely irreversible event because cell survival on selection for either drug resistance or increased metastatic potential may require reexpression of caveolin-1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Swiss National Science Foundation
Grants 3100-050888.97 (to A. F. G. Q.) and 3100-050713.97 (to
C. B.), Swiss Cancer League Grant 636-2-1998 (to A. F. G. Q.),
Chilean National Science Foundation Fondecyt 1990893 (to
A. F. G. Q.) and Europroteome SA (Geneva, Switzerland). 
2 To whom requests for reprints should be
addressed. Present address: Instituto de Ciencias Biomedicas, Facultad
de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Phone/Fax: 56-2-7382015; E-mail: aquest{at}machi.med.uchile.cl
3 The abbreviations used are: MAPK,
mitogen-activated protein kinase; IPTG,
isopropyl-1-thio-ß-D-galactopyranoside; ISREC, Swiss
Institute for Cancer Research; MDCK, Madin-Darby canine kidney.
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subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J. Biol. Chem., 271: 29182-29190, 1996.-isoform of caveolin is selectively phosphorylated by v-Src in vivo. J. Biol. Chem., 271: 3863-3868, 1996. expression by IL-1, tumor necrosis factor, and IL-2. Complex regulation via elements in the 5' flanking region. J. Immunol., 145: 3340-3347, 1990.[Abstract]
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