From the a Department of Molecular Pharmacology and the c Departments of Developmental and Molecular Biology and Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, the e Cancer Therapy and Research Center, San Antonio, Texas 78229, and the g Department of Pathology, McMaster University, West Hamilton, Ontario L8S 4K1, Canada
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ABSTRACT |
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Neu (c-erbB2) is a proto-oncogene product that encodes an epidermal growth factor-like receptor tyrosine kinase. Amplification of wild-type c-Neu and mutational activation of Neu (Neu T) have been implicated in oncogenic transformation of cultured fibroblasts and mammary tumorigenesis in vivo. Here, we examine the relationship between Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Recent studies have suggested that caveolins may function as negative regulators of signal transduction. Our current results show that mutational activation of c-Neu down-regulates caveolin-1 protein expression, but not caveolin-2, in cultured NIH 3T3 and Rat 1 cells. Conversely, recombinant overexpression of caveolin-1 blocks Neu-mediated signal transduction in vivo. These results suggest a reciprocal relationship between c-Neu tyrosine kinase activity and caveolin-1 protein expression. We next analyzed a variety of caveolin-1 deletion mutants to map this caveolin-1-dependent inhibitory activity to a given region of the caveolin-1 molecule. Results from this mutational analysis show that this functional in vivo inhibitory activity is contained within caveolin-1 residues 32-95. In accordance with these in vivo studies, a 20-amino acid peptide derived from this region (the caveolin-1 scaffolding domain) was sufficient to inhibit Neu autophosphorylation in an in vitro kinase assay. To further confirm or refute the relevance of our findings in vivo, we next examined the expression levels of caveolin-1 in mammary tumors derived from c-Neu transgenic mice. Our results indicate that dramatic reduction of caveolin-1 expression occurs in mammary tumors derived from c-Neu-expressing transgenic mice and other transgenic mice expressing downstream effectors of Neu-mediated signal transduction, such as Src and Ras. Taken together, our data suggest that a novel form of reciprocal negative regulation exists between c-Neu and caveolin-1.
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INTRODUCTION |
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Development of the normal breast and breast carcinogenesis involves a complex interplay among growth factors, steroids, activation of oncogenes, and inactivation of tumor suppressor genes (1). The Neu (c-erbB2) proto-oncogene encodes a tyrosine kinase receptor that is amplified and overexpressed in a significant proportion of human breast tumors (1, 2). Increased amplification of the Neu oncogene directly correlates with poor clinical outcome for breast cancer patients in whom the cancer has not spread to the lymph nodes (3, 4).
Neu is a member of a growth factor receptor family that includes the epidermal growth factor (EGF)1 receptor (EGFR),and Neu is therefore also known as HER2, for human EGF receptor 2. The ligands for the Neu receptor are distinct from the structurally related EGFR (5); however, the EGFR is capable of dimerizing with Neu, and this heterodimer can form when only one member of the pair binds ligand (5). Both Neu and the EGFR stimulate proliferation of breast cancer cells, and overexpression of these two proteins is roughly correlated with progression of human breast cancer (6, 7).
Oncogenic activation by Neu can occur through (i) overexpression, (ii) a point mutation within the transmembrane domain, or (iii) small deletions of the membrane-proximal region of the extracellular domain (8). A variety of mutations within the transmembrane domain of the coding sequence of the rat Neu gene (for example, Neu T) are highly oncogenic (8). Overexpression of Neu T in transgenic mice under the control of the MMTV long terminal repeat induces mammary adenocarcinomas with high frequency (9, 10), and overexpression of wild-type Neu can also induce breast tumors (11). Several signaling pathways are activated by Neu, including activation of the Ras GTPase-activating protein and the tyrosine kinase c-Src.
Here, we examine the relationship between Neu tyrosine kinase activity and caveolin-1 protein expression using a variety of in vitro, in vivo, and whole animal approaches. Taken together, our results indicate (i) that increases in Neu tyrosine kinase activity down-regulate caveolin-1 protein expression and (ii) that caveolin-1 overexpression can inhibit Neu-mediated signal transduction. These results suggest that a novel form of reciprocal negative regulation exists between c-Neu and caveolin-1. This is the first in vivo demonstration that caveolin-1 expression can functionally suppress signal transduction from a growth factor receptor to the nucleus.
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EXPERIMENTAL PROCEDURES |
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Materials-- Antibodies and their sources were as follows: anti-caveolin-1 IgG (mAb 2297; gift of Dr. John R. Glenney, Transduction Laboratories, Inc.; for immunoblotting) (12); anti-caveolin-1 (polyclonal antibody; rabbit anti-peptide antibody directed against caveolin-1 residues 2-21; Santa Cruz Biotechnology, Inc.; for immunoblotting and immunocytochemistry), and anti-caveolin-2 (mAb 65; Transduction Laboratories, Inc.) (13). A variety of other reagents were purchased commercially: fetal bovine serum (JRH Biosciences), pre-stained protein markers (Life Technologies, Inc.), and Slow-Fade anti-fade reagent (Molecular Probes, Inc., Eugene, OR). The PathDetectTM Elk-1 in vivo trans-Reporting System was from Stratagene. NIH 3T3 and Rat 1 cell lines expressing c-Neu or mutationally activated Neu were as described previously (14, 15). NIH 3T3 cells expressing wild-type c-Neu or mutants of c-Neu were the generous gift of Dr. Mien-Chie Hung (M.D. Anderson Cancer Center, Houston, TX). NIH 3T3 cells expressing v-Src were the generous gift of Dr. David Shalloway (Cornell University, Ithaca, NY). CHO cells (GRC+ LR-73) were the generous gift of Dr. Jeffrey Pollard and were as described previously (16). Anti-tubulin antibodies were the generous gift of Dr. Lester Binder (Northwestern University Medical School, Chicago, IL) (17).
Cell Culture-- NIH 3T3, Rat 1, and CHO cells were propagated in T-75 tissue culture flasks in Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% serum.
Ceramide Treatment-- Ceramide derivatives (C6-D-erythro, C6-D-erythro-(dihydro), and C6-D-threo) were purchased from Calbiochem and Matreya, Inc. Briefly, NIH 3T3 cells were cultured for 48 h in the presence of a given ceramide derivative (40 µM each) in normal medium (i.e. Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% donor calf serum).
Caveolin-1 Expression Vectors-- Canine caveolin-1 (full-length and deletion mutants) was subcloned into the multiple cloning site (HindIII/BamHI) of the vector pCB7 (containing the cytomegalovirus promoter and a hygroR marker; gift of J. Casanova, Massachusetts General Hospital) for expression in CHO cells.
Assay for Neu-mediated Signal Transduction-- To measure Neu-mediated signal transduction, we employed the PathDetectTM Elk trans-Reporting System. This assay employs a fusion protein that contains the DNA-binding domain of GAL4 and the transactivation domain of Elk to induce expression of a luciferase reporter driven by an artificial promoter containing five GAL4-binding sites. Phosphorylation of the transactivation domain of Elk, in turn, activates transcription of the luciferase gene from the reporter plasmid. Experiments testing a plasmid encoding only the GAL4 DNA-binding domain demonstrated that luciferase expression is specifically dependent on activation of the Elk transactivation domain (data not shown).Transient transfections were performed using calcium phosphate precipitation. Briefly, 300,000 CHO cells were seeded in six-well plates 12-24 h before the transfection. Each point was transfected with 1 µg of pSV, pSV-Neu wt, or pSV-Neu T; 1 µg of pFR-Luc; 50 ng of pFA-Elk (as described by Stratagene); and 1 µg of the indicated caveolin or empty vector. 12 h post-transfection, the cells were rinsed twice with phosphate-buffered saline and incubated in 1% fetal bovine serum for another 24-36 h. The cells were then in lysed in 200 µl of extraction buffer, 50 µl of which was used to measure luciferase activity, as described (18). These assays were made possible through the use of a special CHO-derived cell line called GRC+ LR-73. Unlike parental CHO cells, GRC+ LR-73 cells are a non-transformed growth control revertant that has normal fibroblastic morphology, does not grow in suspension, requires high serum concentrations for growth, and undergoes synchronized growth arrest in low concentrations of serum (1-2%) without a loss of viability (16). Also, these cells have a much higher transfection efficiency (~10-fold) than parental CHO cells.
Immunoblotting-- Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After transfer, nitrocellulose sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting. For immunoblotting, incubation conditions were as described by the manufacturer (Amersham Pharmacia Biotech), except we supplemented our blocking solution with both 1% bovine serum albumin and 2% nonfat dry milk (Carnation).
Northern Analysis-- Total RNA was extracted and purified according to the manufacturer's instructions (QIAGEN Inc.). 10 µg of total cellular RNA was denatured with formaldehyde and subjected to Northern blot analysis with 32P-labeled probes for the mouse caveolin-1 mRNA (2.4 kilobases) and the human 18 S rRNA, as a control for equal loading.
Neu in Vitro Kinase Assay--
A rabbit anti-peptide antibody
directed against Neu C-terminal residues 1169-1186 (C-18; Santa Cruz
Biotechnology, Inc.) was used to immunoprecipitate Neu from CHO cells
transiently transfected with the c-Neu cDNA. In vitro
kinase assays were performed essentially as described previously (19).
Briefly, immunoprecipitates were equilibrated with kinase reaction
buffer (20 mM Hepes, pH 7.4, 5 mM
MgCl2, and 1 mM MnCl2), and the
reaction was initiated by addition of 15 µCi of
[-32P]ATP. After 15 min of incubation at 25 °C, the
reaction was terminated by addition of 2× SDS-polyacrylamide gel
electrophoresis sample buffer and boiling for 2 min. Phosphorylated
c-Neu was detected by autoradiography using an intensifying screen.
Prior to initiating the reaction, immunoprecipitates were preincubated
in kinase reaction buffer with the indicated caveolin-derived peptides
for 1 h at 4 °C. Peptides were dissolved in
Me2SO, and 100× stock solutions were prepared.
Controls omitting peptide contained an equivalent volume of
Me2SO, which did not exceed 1%.
Mouse Models of Mammary Tumorigenesis-- Transgenic mice expressing MMTV-Neu (11), MMTV-Src (20), MMTV-Ras, and MMTV-Myc (21) were as described previously.
Immunohistochemistry-- Paraffin-embedded sections were stained as detailed in a protocol provided by Transduction Laboratories, Inc. Briefly, after re-hydration, slides were placed face up in an incubation container, and each section was covered with a solution of 1% SDS in Tris-buffered saline (100 mM Tris, pH 7.4, 138 mM NaCl, and 27 mM KCl). After a blocking step, sections were incubated with anti-caveolin-1 IgG (1:500 dilution in blocking buffer; Santa Cruz Biotechnology, Inc.) for 2 h at 37 °C. After rinsing with Tris-buffered saline (3 × 5 min each), sections were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200 dilution in blocking buffer; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at 37 °C. After rinsing, coverslips were mounted with Slow-Fade anti-fade reagent, and slides were examined by fluorescence microscopy. Note that pretreatment of sections with 1% SDS has recently been shown to be an effective method for antigen retrieval, especially with anti-caveolin IgG (22).
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RESULTS |
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Mutational Activation of c-Neu Down-regulates Caveolin-1, but Not Caveolin-2, in Cultured NIH 3T3 and Rat 1 Cells-- A variety of Neu constructions that were stably expressed in cultured fibroblasts (NIH 3T3 or Rat 1 cells) are shown in Fig. 1. Note that these include wild-type c-Neu, Neu T (a point mutation within the transmembrane domain), Neu T with a C-terminal deletion, and a series of N-terminal domain deletion mutants. All of these mutational changes are known to greatly increase their oncogenic potential and basal tyrosine kinase activity, as compared with wild-type c-Neu (14, 15). However, Neu T with a C-terminal deletion lacks the ability to undergo autophosphorylation and does not form a stable complex with Shc, yet it is still more active than wild-type c-Neu.
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Recombinant Overexpression of Caveolin-1 Blocks Neu-mediated Signal Transduction in Vivo-- Why is caveolin-1 down-regulated in response to oncogenic Neu? One possibility is that caveolin-1 functions as a negative regulator of Neu-mediated signal transduction. To test this hypothesis of "reciprocal regulation," we employed an assay to measure Neu-mediated signal transduction from the plasma membrane to the nucleus. In this assay, transient coexpression of Neu with a luciferase reporter plasmid allowed us to measure activation of the transcription factor Elk. This luciferase reporter plasmid contains a promoter specifically responsive to Elk phosphorylation and activation (see "Experimental Procedures"). Elk is activated in response to a wide variety of signals, including activation of p42/44 MAP kinase, p38 MAP kinase, and Jun kinase cascades.
As expected, Neu T was ~3.5-fold more active than wild-type c-Neu in this assay system (Fig. 4). However, coexpression of caveolin-1 dramatically inhibited Neu-mediated signal transduction evoked by overexpression of either wild-type c-Neu or mutationally activated Neu T by ~10-20-fold (Fig. 4). Importantly, the empty vector used to express caveolin-1 had no effect by itself. Additionally, overexpression of
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Defining a Region of Caveolin-1 That Inhibits Neu-mediated Signal
Transduction by Deletion Mutagenesis--
We next employed this assay
system and a variety of caveolin-1 deletion mutants to map this
caveolin-1-dependent inhibitory activity to a given region
of the caveolin-1 molecule. The caveolin-1 deletion mutants that were
used are shown schematically in Fig. 5A. Note that the full-length
caveolin-1 molecule contains residues 1-178 (-isoform), the
-isoform lacks residues 1-31,
C lacks residues 141-178 of the
C-terminal domain, and
N lacks residues 1-95 of the N-terminal
domain. These constructs have all been previously characterized and are
expressed to equivalent levels in transfected cells (25). Fig.
5B shows the results of this mutational analysis. Virtually
identical results were obtained for both wild-type c-Neu and
mutationally activated Neu T. Relative to wild-type full-length
caveolin-1 (FL;
-isoform),
C was ~10-20-fold more
potent, and
N was ~10-fold less potent; the
-isoform was almost
as potent as wild-type full-length caveolin-1. From this mutational
analysis, we can conclude that the in vivo inhibitory activity of caveolin-1 is contained within the N-terminal domain and,
to a first approximation, within caveolin-1 residues 32-95.
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Peptides Derived from the Caveolin Scaffolding Domain Inhibit Neu Tyrosine Kinase Activity in Vitro-- One possible explanation for the ability of caveolin-1 to impede Neu-mediated signal transduction is that caveolin-1 interacts directly with the Neu tyrosine kinase. We decided to test this hypothesis by examining the effects of synthetic peptides derived from caveolin-1 on the in vitro kinase activity of c-Neu. Fig. 6A shows that the antibody used for these experiments efficiently immunoprecipitated recombinantly expressed c-Neu. The potential inhibitory activity of caveolin-1-derived peptides was evaluated using an in vitro kinase assay that measures the activity of immunopurified recombinant wild-type c-Neu (Fig. 6, B and C). Only the caveolin-1 peptide encoding caveolin-1 residues 61-101 or 82-101 showed significant inhibitory activity, whereas a peptide encoding an adjacent region of caveolin-1 (residues 53-81) had no effect at the same concentration. Note that residues 82-101 correspond to the scaffolding domain (SD) of caveolin-1. However, if the peptide encoding caveolin-1 residues 82-101 was divided into two shorter peptides (residues 84-92 and 93-101), its inhibitory activity was abolished.
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Expression of Caveolin-1 in Normal Mouse Mammary Tissue and Neu-induced Mammary Tumors-- The Neu (c-erbB2) proto-oncogene is amplified and overexpressed in a significant proportion of human mammary tumors (1, 2). Are our current findings relevant in whole animals and specifically to mammary tumorigenesis? To answer this question, we chose to evaluate the expression of caveolin-1 protein in normal mouse mammary tissue and in mammary tumors from an established mouse model of c-Neu-induced mammary tumorigenesis. Targeted overexpression is achieved by using the MMTV long terminal repeat to drive mammary-specific expression of a given oncogene. Using this strategy, mammary tumorigenesis can be induced by targeted overexpression of several different classes of oncogenes, including the c-Neu receptor tyrosine kinases (9), cytoplasmic tyrosine kinases (Src) (20), nuclear transcription factors (Myc) (21), and cytoplasmic GTPases (Ras) (28).
Fig. 7 shows a Western blot analysis of the expression of caveolin-1 protein in 10 independent mammary tumors, each derived from a different MMTV-Neu transgenic mouse. Normal virgin mammary tissue served as positive controls for caveolin-1 expression. Immunoblotting with anti-tubulin antibodies served as a control for equal loading. Note that loss or dramatic reduction of caveolin-1 expression occurred in mammary tumors derived from several independent c-Neu-expressing transgenic lines.
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What About Downstream Effectors of the Neu Signaling Pathway?-- Src family tyrosine kinases and Ras are thought to be downstream components of the Neu signaling pathway and have also been implicated in mammary tumorigenesis. Thus, we next examined caveolin-1 expression in mammary tumors from mice transgenically expressing MMTV-Src and MMTV-Ras. Fig. 8 shows that as with MMTV-Neu tumors, caveolin-1 expression was also dramatically reduced or undetectable in these tumors as well. Also, virtually identical results were obtained with tumors from MMTV-Myc mice. The histopathological appearances of these 25 independent mammary tumors included four distinct types of mammary tumors: comedo, scirrhous, papillary, and acinar adenocarcinomas.
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Immunolocalization of Caveolin-1 in Normal Mouse Mammary Tissue and Mammary Tumors-- Mammary tissue consists of a complex array of cells, including the mammary epithelium, fibroblasts, adipocytes, endothelial cells, smooth muscle cells, and many other cell types. However, it remains unknown whether caveolin-1 is expressed in normal mammary epithelium. Caveolin-1 expression has been well documented in fibroblasts, adipocytes, endothelial cells, and smooth muscle cells. Thus, "down-regulation" of caveolin-1 expression observed by Western analysis may simply reflect tumor mass derived from mammary epithelium that normally lacks caveolin-1 expression.
To examine this possibility, we immunolocalized caveolin-1 within normal mammary tissue. Our results indicate that caveolin-1 is normally abundant in the mammary epithelial cells, as the alveolar ductal epithelia were quite brightly stained with an anti-caveolin-1 antibody probe (Fig. 9). We next examined the distribution of caveolin-1 expression in mammary tumors derived from all four transgenic mouse models of mammary tumorigenesis (Fig. 9). In all cases, little or no caveolin-1 staining of the tumor mass itself was observed. However, adjacent fibro-fatty connective tissue remained caveolin-1-positive, and the vasculature within the tumors (consisting of endothelial cells and smooth muscle cells) was also brightly stained. These results clearly show that caveolin-1 is abundantly expressed in normal mammary epithelium, but that cancerous transformed mammary tumor cells have lost caveolin-1 expression.
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DISCUSSION |
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Caveolae are plasma membrane-attached vesicular organelles with a diameter of ~50-100 nm (29, 30). It has been proposed that caveolae play a pivotal role in a number of essential cellular functions, including signal transduction, lipid metabolism, cellular growth control, and apoptotic cell death. The principal protein components of caveolae are the caveolin family of proteins, termed caveolin-1, -2, and -3 (29, 30). Caveolins provide a means for caveolin-interacting proteins to be concentrated within caveolae membranes. Caveolins interact directly with a number of caveolae-associated signaling molecules, such as Ha-Ras, heterotrimeric G-proteins, epidermal growth factor receptor, protein kinase C, Src family tyrosine kinases, and nitric-oxide synthase isoforms (19, 26, 27, 31, 32). In many of these cases, it has been documented that caveolin binding can effectively inhibit the enzymatic activity of these signaling molecules in vitro.
Three caveolin genes have been identified thus far: Cav-1,
Cav-2, and Cav-3. Caveolin-1 mRNA gives rise
to two isoforms via alternate translation initiation from methionines 1 and 32, resulting in caveolin-1 (residues 1-178) and caveolin-1
(residues 32-178) (12). Caveolin-1 and -2 appear to be most highly
expressed in endothelial cells and adipocytes (13, 33). The expression of caveolin-3 appears to be confined to striated and smooth muscle cell
types (34-36).
As caveolin-1 was the first caveolin family member to be identified, it has served as the prototype for the study of caveolins and caveolae function. Caveolin-1 is a 22-24-kDa integral membrane protein that consists of 178 amino acid residues. The central region of caveolin-1 contains a string of 33 hydrophobic amino acids that function as a membrane anchor, allowing caveolin-1 to assume a hairpin configuration with both N- and C-terminal domains facing the cytoplasm. Residues 61-100 of caveolin-1 function to direct the self-oligomerization of caveolin-1 and as a plasma membrane-bound scaffold to sequester specific caveolin-interacting proteins within caveolae membranes (19, 37, 38).
Here, we show that mutational activation of c-Neu down-regulates caveolin-1 protein expression, but not caveolin-2, in cultured NIH 3T3 and Rat 1 cells. A dramatic reduction of caveolin-1 expression also occurs in mammary tumors derived from c-Neu-expressing transgenic mice and other transgenic mice expressing downstream effectors of Neu-mediated signal transduction. Conversely, recombinant overexpression of caveolin-1 blocks Neu-mediated signal transduction in vivo. Results from this mutational analysis show that this in vivo inhibitory activity is contained within caveolin-1 residues 32-95. Furthermore, a specific peptide encoding the caveolin-1 scaffolding domain (residues 82-101) inhibits Neu tyrosine kinase activity in vitro. These results suggest a reciprocal relationship between c-Neu tyrosine kinase activity and caveolin-1 protein expression.
Our present results are consistent with a number of independent experimental observations. (i) Transformation of NIH 3T3 cells with oncogenic forms of Abl or Ras is sufficient to down-regulate caveolin-1 protein expression; however, caveolin-2 protein levels remain unchanged (13, 24). (ii) A variety of receptor tyrosine kinases (platelet-derived growth factor receptor, insulin receptor, nerve growth factor receptor (trk), EGFR, and c-Neu) have been shown to co-fractionate with caveolin-1 in a variety of cell fractionation approaches (26, 39-43). (iii) Caveolin-1 can interact directly with the EGFR in vitro, through the recognition of a conserved caveolin-binding motif within the kinase domain; this interaction is sufficient to inhibit the in vitro autophosphorylation of the purified EGFR kinase domain (26). However, the in vivo relevance of these results has remained unknown. In this study, we have demonstrated that caveolin-1 inhibits Neu-mediated signaling in vivo and in vitro. As Neu possesses a putative caveolin-1-binding motif within its kinase domain that is identical to that of the EGFR (DVWSYGVTVWEL in both c-Neu and the EGFR), it is not unreasonable to suspect a direct interaction between c-Neu and caveolin-1.
Of note, we observed an interesting difference between the effects of c-Neu overexpression in cultured cells and in transgenic mice. In cultured Rat 1 cells, overexpression of wild-type c-Neu was not sufficient to effectively down-regulate caveolin-1. In striking contrast, targeted expression of wild-type c-Neu in mouse mammary cells was sufficient to down-regulate caveolin-1 in mammary tumors. These results suggest that mammary cells in vivo may be more sensitive to the down-regulation of caveolin-1. Alternatively, c-Neu may have undergone somatic mutations during the process of mammary tumorigenesis, as has been documented previously (8). In addition, these findings point out that, in some instances, results with cultured cells may not adequately reflect the in vivo situation.
This is particularly relevant in the case of caveolin-1, as many cell lines that we have examined lack caveolin-1 expression, whereas caveolin-1 is well expressed in the corresponding tissue in vivo. In many instances, these cell lines were derived from the primary culture of tumors, suggesting that caveolin-1 expression may be very sensitive to down-regulation during cell transformation. However, more examples will be necessary before we can conclude that caveolin-1 down-regulation is a general response to any form of cell transformation.
As amplification or mutational activation of Neu occurs in humans and has been implicated in the development of human breast cancers, our current experimental observations may have implications for the future diagnosis or treatment of human breast cancers. For example, it will be important to examine the possible relationship among c-Neu amplification/activation, caveolin-1 down-regulation, and clinical outcome in patients with breast cancers.
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ACKNOWLEDGEMENTS |
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We thank members of the Pestell and Lisanti laboratories for insightful discussions and Dr. Jeffrey Pollard for critical reading of the manuscript and donating the CHO cell line.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health FIRST Award GM-50443 and by the G. Harold and Leila Y. Mathers Charitable Foundation and the Charles E. Culpeper Foundation (all to M. P. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Supported by National Institutes of Health Medical Scientist Training Program Grant T32-GM07288.
d Supported by Cancer Center Core Grant 5-P30-CA13330-26 from the National Institutes of Health.
f Supported by Grant DHP-150 from the American Cancer Society.
h Supported by research grants from the Canadian Breast Cancer Initiative and recipient of a Medical Research Council of Canada scientist award.
i Supported by National Institutes of Health Grants R29-CA70897 and R01-CA75503 and by an Irma T. Hirshl/Susan Komen Award.
j To whom correspondence should be addressed: Dept. of Molecular Pharmacology and the Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti{at}aecom.yu.edu.
The abbreviations used are: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; CHO, Chinese hamster ovary; MMTV, murine mammary tumor virus; MAP, mitogen-activated protein kinase.
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REFERENCES |
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