Download Invasion Activating Caveolin-1 Mutation in Human Scirrhous Breast

[CANCER RESEARCH 61, 2361–2364, March 15, 2001]
Advances in Brief
Invasion Activating Caveolin-1 Mutation in Human Scirrhous Breast Cancers1
Kazuhiko Hayashi,2 Satoru Matsuda,2, 3 Kazuya Machida, Tatsuyoshi Yamamoto, Yoshihide Fukuda, Yuji Nimura,
Tetsuo Hayakawa, and Michinari Hamaguchi
Department of Molecular Pathogenesis [K. H., S. M., K. M., M. H.], Second Department of Internal Medicine [K. H., K. M., Y. F., T. H.], and First Department of Surgery [T. Y.,
Y. N.], Nagoya University School of Medicine, Nagoya 466-8550 Japan
Abstract
We looked for mutations in the caveolin-1 gene, encoding a critical
molecule for membrane signaling to cell growth, in 92 primary human
breast cancers, and we report here the identification of a mutation in
caveolin-1 at codon 132 (P132L) in 16% of cases. The mutation-positive
cases were mostly invasive scirrhous carcinomas. In cell lines expressing
the same mutant of caveolin-1, we observed that the mutant Caveolin-1
expression seemed to induce cellular transformation and activation of
mitogen-activated protein kinase-signaling pathway and to promote invasion-ability as well as altered actin networks in the cells. These results
provide, for the first time, genetic evidence that a functioning Caveolin-1
mutation may have a role in the malignant progression of human breast
cancer.
Introduction
Molecular cloning had identified three distinct caveolin genes
(1– 4) caveolin-1, caveolin-2, and caveolin-3. Alignment of the protein sequences encoded by these caveolin genes is shown in Fig. 1a.
Note that most of the predicted functional domains of the caveolins
are well-conserved among the family members, including the scaffolding domain and the membrane-spanning domain. It is well-known
that one of the conserved amino acids in caveolin-3, the expression of
which is muscle-specific, was found to be a disease-involved site
(5– 8). This site is evolutionarily conserved from worms to man,
providing evidence that this region of the membrane-spanning domain
is critical for Caveolin function. From these exciting findings, we
surmised that the mutation of the amino acid in this site might
function in an alternative physiological role among the caveolins.
Then, we targeted the site to survey mutations in caveolin-1 in human
tumors, because previous reports suggest identifying caveolin-1 as a
candidate tumor suppressor gene (9 –11).
Materials and Methods
Preparation and Analysis of DNA and RNA. We extracted DNA from
primary tumors, corresponding noncancerous tissues, cultured cells, and blood
leukocytes, as described before (12). Total RNAs were also isolated with
Trizol Reagent (Life Technologies, Inc.) according to the manufacturer’s
protocol. Five ␮l (1–3 ␮g/␮l) of mRNA were reverse transcribed to generate
cDNA using Superscript II and random hexamers (Life Technologies, Inc.)
according to manufacturers’ protocols.
PCR-RFLP Analysis. The Ucav (sense, 5⬘-TTGGAAGGCCAGCTTCAC-3⬘) and Dcav (antisense, 5⬘-GATAGGAACTTTACAGT-3⬘) PCR
Received 11/21/00; accepted 1/25/01.
The costs of publication of this article were defrayed in part by the payment of page
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1
Supported by a Grant-in-Aid for scientific research on priority areas and for COE
Research from the Ministry of Education, Science, and Culture of Japan and by a grant
under the Monbusho International Scientific Research Program.
2
K. H. and S. M. contributed equally to this work.
3
To whom requests for reprints should be addressed, at Nagoya University School of
Medicine, Department of Molecular Pathogenesis, 65 Tsurumai-cho, Showa-ku, Nagoya,
Japan, 466-8550. Phone: 81-52-744-2463; Fax: 81-52-744-2464; E-mail: smatsuda@
med.nagoya-u.ac.jp.
primers were designed specifically to amplify the caveolin-1 sequences. The
amplified caveolin-1 DNA fragments from genomic DNAs or cDNAs were
digested at 37°C for 5 h with the indicated restriction endonucleases. The
digested DNAs were electrophoresed in 0.8% agarose gel or in 6% polyacrylamide gel before the UV-photos.
Tissue Samples, Cell Lines, and Antibodies. We obtained tumors and
corresponding normal breast tissues with informed consent from 92 patients
who had undergone mastectomy. All tumors were diagnosed histopathologically as carcinomas. Parental NIH3T3 cells were transfected, with the mutant
or wild-type caveolin-1 genes introduced into pcDNA3 expression vector using
a lipofection protocol. Resistant clones were selected with G418, as described
before. Antibodies used were as follows: (a) antihuman caveolin-1, antihuman,
and antimouse caveolin-1 (Transduction Laboratories); and (b) anti-Ras, antiphosphoMAPK, anti-ERK2, anti-phopho-p38, anti-p38, anti-phophoAKT, and
anti-AKT (New England Biolaboratories).
Sequence Analysis. We purified aberrant PCR products detected by PCRRFLP study. The DNA sequences of each aberrant sample and some normal
samples were determined (13) using an Applied Biosystems DNA sequencer
with a Dye-terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). We also confirmed all mutations by repeated experiments (at least
three times) using DNAs extracted from the tumor and corresponding tissues.
Cell Migration Assay. Cells were grown in the presence of 10% fetal
bovine serum until confluence. A wound area was generated by scraping with
a plastic scraper. After 2 days, cells in the wounded monolayer were counted
randomly at multiple fields.
Invasion Assay. Cells were assayed for their invasiveness by a modified
Boyden chamber method. Briefly, conditioned media obtained from NIH3T3
were placed in the lower compartment of the chamber. Cells suspended in
serum-free DMEM were seeded onto Matrigel-coated filters. After 12 h of
incubation, cells that had invaded to the lower surface of the filter were fixed,
stained, and counted.
Immunofluorescence and Western Blotting. To visualize polymerized
actin, cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with
0.5% Triton X-100 for 10 min, and incubated with 1 ␮g/ml FITC-labeled
phalloidin (Sigma Chemical Comp.) for 1 h. Antibody against actin (Sigma
Chemical Co.) also was used for indirect immunofluorescence. For the immunofluorescence, cells were also fixed with methanol/acetone for 10 min and
permeabilized with 0.5% Triton X-100 for 10 min. They were incubated with
the indicated antibody for 1 h at 37°C before a 1-h incubation with fluorescein
isothiocyanate-conjugated goat anti-IgG antibody (Sigma Chemical Co.). Cells
were viewed on a Nikon microscope and photographed. Western blotting was
carried out according to the methods described before (12).
Results and Discussion
To assess the potential for caveolin-1 mutation in cases of breast
cancer, we constructed PCR primers to specifically amplify a DNA
fragment of human caveolin-1 sequences from genomic DNA of the
tissue. We used conventional PCR-RFLP mutational screening because RFLP is a reliable and easy method for mutation screening if a
proper enzymatic site exists in the target region (14). As can be seen
in Fig. 1b, the mutation in codon 132 eliminated Rsa-I and/or Nla III
sites; then we were able to develop a rapid screen for the caveolin-1
mutation using Rsa-I- and Nla III-based RFLP assay. After purifying
genomic DNAs derived from paired normal and tumor samples, we
amplified caveolin-1 DNA fragments using the specific primers and
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A CAVEOLIN-1 MUTATION ACTIVATES INVASION OF THE CELLS
Fig. 1. Alignment of the protein sequences encoded by the human caveolin-1, caveolin-2, and caveolin-3. a, conserved amino acid residues are boxed. A Pro-Leu substitution
at amino acid residue 105 in human caveolin-3 that led to an autosomal dominant form of
limb-girdle muscular dystrophy is indicated by an ⴱ. Dashed-underlined amino acids and
underlined amino acids correspond to the scaffolding domain and the hydrophobic
membrane-spanning region, respectively. b, DNA sequence and restriction enzyme sites
corresponding to around the Pro-132 in caveolin-1 are shown. Note that both Rsa-I and
Nla-III are available and necessary for detecting the DNA-mutation corresponding to the
Pro-132.
Fig. 3. Identification of caveolin-1 mutations in human breast cancer specimen. a,
DNA sequencing electropherograms of the region consisting of Rsa-I and Nla-III sites
depicting the mutation. Although the genomic mutation is heterozygous, a manual
sequencing result derived from cloned PCR products is shown. Arrowhead, the site of
mutation. Histopathological evaluation of the carcinomas is also shown in parentheses.
Every sequence was verified several times by independent sequencing. b, DNA sequence
and the mutation sites corresponding to the Pro-132 and Cys-133 in caveolin-1 are shown.
c, representative direct DNA sequencing chromograms corresponding to the mutation site.
The mutation is at codon 132, leading to Leu instead of to Pro.
Fig. 2. a– c, a representative result of PCR-RFLP analyses in breast cancer tissues (T)
and the paired normal tissues (N) for detecting mutation. Polyacrylamide gels stained with
ethidium bromide are shown and fragment lengths are given in bp on the right. a, Nla-III
digestion. Heterozygous mutants are shown by ⴱ. b, Hinf-I digestion. No mutation is
detected. c, Rsa-I digestion. No mutation is detected. d–f, a representative result of
PCR-RFLP analyses in blood leukocytes from healthy volunteers. Neither mutation nor
deletion is detected in every panel. d, no digestion. e, Rsa-I digestion and Nla-III
digestion. f, Hinf-I digestion.
performed PCR-RFLP assays (Fig. 2, a– c). First, we screened DNA
from 92 primary breast cancers for alterations of caveolin-1 and found
15 genetic alterations (7 scirrhous carcinomas, 3 solid tubular carcinomas, 2 papillo-tubular carcinomas, 2 invasive-lobular carcinomas,
and 1 unknown carcinoma) among them. Almost all of the mutationpositive cases were invasive and/or scirrhous carcinomas. Mutations
were confirmed by manual sequencing analyses (Fig. 3). Pilot studies
using the PCR-RFLP identified two cases (scirrhous carcinoma) of
double mutations in codon 132 and 133 that were confirmed by
manual sequencing (Fig. 3, a and b), but we were unable to determine
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A CAVEOLIN-1 MUTATION ACTIVATES INVASION OF THE CELLS
Fig. 4. Derivation and characterization of NIH3T3 cells harboring the caveolin-1 mutation. a, expression of the exogenous human caveolin-1 mutant in normal mouse NIH3T3 cells
by Western blot analysis. Lysates were prepared from parental NIH3T3 cells (3T3) and NIH3T3 cells harboring the P-L mutation (P132L) of caveolin-1 (termed Cl3, Cl4, Cl5, and
Cl6). Immunoblotting was performed with antihuman caveolin-1 (h-Cav1) and antimouse (t-Cav1). Western blots using anti-Ras- and anti-SHPS1-specific antibodies are also shown
as controls. In all panels, each Lane contains an equal amount of total protein. b, parental NIH3T3 cells (3T3) and NIH3T3 cells harboring the P-L mutation of caveolin-1 (Cl3) were
compared morphologically. c, parental NIH3T3 cells (3T3) and NIH3T3 cells harboring the P-L mutation of caveolin-1 (Cl3) were compared with immunofluorescence microscopy
of actin stress fibers stained with phalloidin. d, immunoblot analyses with antiphospho-MAPK, antiphospho-p38-MAPK, and antiphospho-AKT. Immunoblots with the antibodies
against the nonphosphorylated kinases are also shown. In all panels, each Lane contains an equal amount of total protein. e–f, invasive ability of the cells. The invasive ability was
assayed by a modified Boyden chamber method. e, average values ⫾ SD of a typical experiment are shown. The result of active v-Src-transformed-3Y1 cells (SR) is also shown as
a positive control. f, a representative result of the invasion assay. Note that numbers of cells penetrated the membrane, which appeared as black spots, in both Cl3 and SR cells compared
with the WT and NIH3T3 control cells. These results were confirmed by several additional experiments and by using more than three independent clones.
whether the mutations were allelic, because RNA from these samples
was not available. Direct DNA sequencing also confirmed the mutation (Fig. 3c). We could not observe loss of heterozygosity of the
normal caveolin-1 gene in any of the breast cancer specimens, and
none of the tumors harboring the mutation demonstrated microsatellite instability (data not shown). Although we screened 92 primary
human breast cancer specimens with this assay, we were unable to
successfully identify the P-L mutation in any of these tumors in DNA
derived from normal, adjacent, matched tissue samples from patients
with tumors harboring the codon 132 mutation. Furthermore, the
genomic DNA analyses of the 26 normal healthy volunteers, from
whom DNA was obtained from their peripheral blood leukocytes,
failed to show that mutation at the site of caveolin-1 (Fig. 2, d–f). In
addition, none of the other eight normal healthy volunteers had such
mutations in the mRNA from their normal tissues (data not shown).
We concluded that all of those mutations found in the breast carcinomas occurred as somatic events.
Taken together, our results and previous findings prompted us to
hypothesize that the mutation at this site in caveolin-1 might lead to
an advantage for tumor growth in the cells. Then, to determine the
transformation potential of caveolin-1, NIH3T3 cells stably transfected with the mutant caveolin-1 (P132L) or wild-type caveolin-1
were derived using a mammalian expression vector. As shown in Fig.
4a, clones Cl3, Cl4, Cl5, and Cl6 were derived, and they expressed
exogenous mutant caveolin-1 at various expression levels, whereas
Ras and SHPS-1 (15) levels were not affected by the expression of the
mutant caveolin-1. Examination of NIH3T3 cells harboring the caveolin-1 mutant by microscopy reveals that these cells have an overall
altered morphology compared with that of cells expressing wild-type
caveolin-1 or parental NIH3T3 (Fig. 4b). All independent caveolin-1
mutant cells had similar morphology, whereas untransfected NIH3T3
cells have an elongated, flattened, spindle-shaped, and stretched morphology. Furthermore, fiber-like phalloidin staining was not observed
with NIH3T3 cells harboring the caveolin-1 mutant, in contrast with
the typical fiber-like appearance of actin cytoskeleton networks with
parental NIH3T3 cell controls (Ref. 16; Fig. 4c). Phalloidin staining of
the other mutant cells of caveolin-1 (Cl4 and Cl5) were similar to that
seen in the Cl3, and staining with anti-actin antibody also showed the
disruption of actin cytoskeleton in the mutant cells (data not shown).
We also examined effects of the caveolin-1 mutant expression on
colony-formation in soft agar to assess anchorage-independent cell
growth and found that these NIH3T3 cells harboring the caveolin-1
mutant exhibited growth in soft agar (parental NIH3T3 and NIH3T3
transfected with vector only showed no colony formation, whereas
NIH3T3 with the caveolin-1 mutant showed ⬎150 colonies/␮g DNA;
n ⫽ 2), in agreement with the previous report showing the dominant
effect of the caveolin-1 mutant (17).
It seemed that the state of tyrosine phosphorylation was not altered
by the expression of the caveolin-1 mutant. We next used a variety of
phospho-specific antibodies (12) that have been generated against the
activated forms of well-known signal transducers. Fig. 4d shows that
both intracellular MAPKs and p38-MAPK4 were constitutively activated in the caveolin-1 mutant clones. On the other hand, the status of
phosphorylation of AKT was not changed, although v-Src-transformed cells (SR3Y1) showed increased phosphorylation of AKT.
4
The abbreviations used are: MAPK, mitogen-activated protein kinase; WT, wild type.
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A CAVEOLIN-1 MUTATION ACTIVATES INVASION OF THE CELLS
Again, these experiments demonstrated the capacity for the mutant to
drive cell transformation. These observations are compatible with
previous reports showing that alteration of caveolin-1 function could
be involved in the cellular transformation as a dominant negative
effect of caveolin-1.
The mutation-positive cases of breast cancer were mostly involved
in the pathologically invasive types such as scirrhous carcinomas (18).
Hence, we suspected that the expression of the caveolin-1 mutant may
affect the invasive ability of the cells.
The invasiveness of cells was then evaluated by the modified
Boyden chamber method as described (19). As can be seen in Fig. 4,
e and f, the mutant cells could penetrate through the reconstituted
membrane to a level similar to that of SR3Y1, whereas the wild-type
caveolin-1 cells and parental NIH3T3 could not. In addition, rapid in
vitro cell motility evaluated using a wound healing assay (20) showed
that the transfectants of the mutant exhibited high motility-potential
compared with the parental cells or with the clones of wild-type
caveolin-1 (data not shown).
Caveolin-1 has been reported to participate in oncogenic processes
in vitro, yet no genetic evidence had been presented that implicated
this gene in the development or the progression of human cancer.
Although Hurlstone et al. (9) reported previously that there was no
mutation in the caveolin-1 gene in human cancers, we sought the
mutation more intensively, focusing on human breast cancers. Here
we have provided evidence for the existence of at least one naturally
occurring mutant form of caveolin-1 that appears to have a role in
human cancer. The results presented in this paper revealed that the
mutation of caveolin-1 had a dominant negative effect on cell transformation and invasiveness. In addition, these findings indicate that
caveolin-1 is likely to function as a tumor suppressor. We speculate
that other effective caveolin-1 mutations, which we have not found
yet, might exist, because there are other consensus sites for caveolin
family members that were found to be at least critical sites in the
scaffolding domain for caveolin-3 in limb-girdle muscular dystrophy.
At this time, the study demonstrated in this paper may provide an
experimental basis for additional analysis of caveolin-1 mutation in
human diseases. In addition, investigation of signaling pathways
affected by caveolins should provide additional insights into the
molecular pathogenetical action of caveolae disorders.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Acknowledgments
We thank Yu-ki Iwata for her excellent technical assistance.
18.
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Research.
Invasion Activating Caveolin-1 Mutation in Human Scirrhous
Breast Cancers
Kazuhiko Hayashi, Satoru Matsuda, Kazuya Machida, et al.
Cancer Res 2001;61:2361-2364.
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