Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA–mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP–dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion. [Cancer Res 2009;69(22):8594–602]
- breast cancer
- lipid rafts
Invadopodia are ventral membrane protrusions that can degrade the extracellular matrix (ECM; ref. 1). A variety of invasive tumor cells, including mammary adenocarcinoma, are reported to form invadopodia when they are cultured on physiologic substrates (2). Invadopodia are thought to function in cancer cell migration in vivo through the physical barriers of the dense ECM existing in the tumor microenvironment, which is a prerequisite for cancer invasion and metastasis (3). Invadopodia are enriched with actin filaments (F-actin) and proteins involved in the actin cytoskeleton, cell signaling, cell-ECM adhesion, and membrane remodeling (4, 5). The matrix degradation activity of invadopodia is mainly mediated by the focal concentration of membrane type 1 matrix metalloproteinase (MT1-MMP) at the surface of invadopodia (6–8). However, the molecular mechanisms underlying the recruitment of invadopodia components are not well understood.
Many plasma membrane proteins are compartmentalized into sphingolipid- and cholesterol-rich microdomains called lipid rafts (9). Lipid rafts are enriched with a variety of proteins, including caveolins and flotillins (10), and thought to function as platforms for localizing signaling proteins and eliciting spatially controlled signal transduction (11). Because lipid raft membranes are actively internalized and trafficked in the cell, lipid rafts are also involved in the transport of lipid raft-associated molecules (12). Recent studies have confirmed the importance of lipid rafts and associated proteins in the pathogenesis of several diseases, including cancer progression (13, 14).
Caveolae are flask-shaped membrane invaginations and major subtypes of lipid rafts (15). Caveolins are integral membrane proteins and major protein components of caveolae and lipid rafts (16). The caveolin family consists of three isoforms in mammals: caveolin (Cav)-1, -2, and -3 (17). Cav-1 is coexpressed with Cav-2 in a variety of tissues, whereas Cav-3 expression is restricted to muscle tissues (17). Genetic studies with knockout mice showed that Cav-1, but not Cav-2, is essential for caveolae formation (15). Cav-1 regulates diverse cellular processes, including raft-mediated endocytosis, vesicular transport, cell migration, and signal transduction (16–19). Clinical studies revealed that Cav-1 upregulation is associated with a poor prognosis and the occurrence of metastatic regions in several human cancers (20, 21). It was recently reported that Cav-1 regulates cell migration and invasion in human breast cancer cells (22). However, the molecular mechanisms by which Cav-1 regulates the ECM remodeling associated with cancer cell invasion are poorly understood.
In this study, the roles of lipid rafts and associated molecules in invadopodia formation are investigated in human breast cancer cells. It is shown that invadopodia are the sites where lipid raft formation and trafficking occur and that Cav-1 is an essential regulator of invadopodia-mediated breast cancer cell invasion.
Materials and Methods
Human breast cancer cell lines MDA-MB-231, T47D, BT549, and Hs578T were obtained from the American Type Culture Collection. MDA-MB-453 and SK-BR-3 were obtained from RIKEN BRC Cell Bank and Cell Resource Center for Biomedical Research Tohoku University, respectively. MDA-MB-231 cells were maintained in a 1:1 mixture of high-glucose DMEM and RPMI 1640 supplemented with 10% fetal bovine serum, 10 units/mL penicillin, and 10 μg/mL streptomycin. Other cell lines were maintained according to the cell banks' instructions.
For Cav-1 constructs, cDNAs encoding human and mouse Cav-1 were amplified by reverse transcription-PCR with total RNA prepared from MDA-MB-231 cells and mouse intestine, respectively. The cDNAs were subcloned into pEGFP-C1, pECFP-C1, and pmCherry-C1 vectors (Clontech). For MT1-MMP constructs, the cDNA clone of human MT1-MMP was obtained from Kazusa DNA Research Institute and subcloned into pEGFP-N1 and pmCherry-N1 vectors (Clontech).
Anti–flotillin-1 (Flot-1), Cav-1, Cav-2, and Cdc42 antibodies were purchased from BD Biosciences. Anti–MT1-MMP antibodies were from Abcam (ab38971) and Daiichi Fine Chemical (114-6G6). Anti-cortactin and β-actin antibodies were from Millipore. Anti–cholera toxin B subunit (CTxB), Src, and Arf6 antibodies were from Calbiochem, Cell Signaling Technology, and Santa Cruz Biotechnology, respectively. Anti–N-WASP antibody was described previously (23).
Fluorescent gelatin-coated coverslips were prepared as described previously (24). Breast cancer cells were cultured on the coverslips for indicated periods. In general, MDA-MB-231 cells begin to degrade the gelatin matrix ∼3 h after plating. In the case of cells overexpressing MT1-MMP, they were cultured on the gelatin matrix for 2 h. To quantitate the degradation activity of invadopodia, 10 randomly selected fields, usually containing 30 to 50 cells, were imaged with a ×60 objective for each determination. The degradation area was determined by using ImageJ 1.41 software and normalized for the number of cells. In each analysis, the mean value of the control cells was set at 100% and the relative values of the cells treated with small interfering RNAs (siRNA) or inhibitors were then calculated. Data are mean ± SE of at least four independent determinations.
Immunofluorescence and time-lapse microscopy
Cells were stained with the indicated antibodies and phalloidin as described previously (23). The cells were observed with an Olympus IX81-ZDC-DSU confocal microscope equipped with a cooled CCD camera (ORCA-ER; Hamamatsu) or with an Olympus confocal laser scanning microscope FV-1000. Time-lapse series of the cells were taken at 37°C using an Olympus IX81-ZDC-DSU microscope equipped with a cooled CCD camera, humidified CO2 chamber, and autofocus system and operated by MetaMorph software (Universal Imaging Corp.).
The stealth RNA interference molecules (Invitrogen) used were Negative Control Medium GC Duplex #2 and Stealth Select RNA interference for Cav-1 (HSS141466 and HSS141467), Cav-2 (HSS101398), and Flot-1 (HSS115568). Cells were transfected with 30 nmol/L siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. The cells were cultured for 48 h and subjected to invadopodia assay or immunoblotting.
Immunoblotting was done as described previously (23). Equal amounts of protein (10-40 μg) were loaded per lane for each analysis. Protein concentration was determined by using BCA Protein Assay kit (Pierce).
Plasmid transfection and retroviral infection
Cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For retroviral infection, cDNAs were inserted into the pMXs-IP vector and recombinant retroviruses were produced with the Plat-A packaging cell line as described previously (25). Cells were then infected with the recombinant retroviruses and selected with 0.5 μg/mL puromycin.
Total RNA was isolated with a RNeasy Mini Kit (Qiagen). Template cDNAs were synthesized with SuperScript III (Invitrogen). PCR amplification was done with KOD plus DNA polymerase (Toyobo). The primer pairs used were as follows: human Cav-1 (5′-ATGTCTGGGGGCAAATACG-3′ and 5′-TTATATTTCTTTCTGCAAGTTGATGC-3′), mouse Cav-1 (5′-ATGTCTGGGGGCAAATACG-3′ and 5′-TCATATCTCTTTCTGCGTGCTG-3′), and human cyclophilin B2 (5′-GCACAGGAGGAAAGAGCATC-3′ and 5′-CTTCTCCACCTCGATCTTGC-3′).
Labeling cells with CTxB
Cells cultured on gelatin-coated coverslips were washed with ice-cold PBS and incubated with 10 μg/mL fluorescent CTxB (Invitrogen) in PBS for 20 min on ice. For the internalization experiments, cells were labeled with CTxB in the growth medium for 20 min on ice and then incubated for 10 min at 37°C. After fixing in 4% formaldehyde for 20 min, the cells were labeled with anti-CTxB antibody in 1% bovine serum albumin and 1% fetal bovine serum in PBS for 1 h at room temperature. For time-lapse imaging, cells were incubated with CTxB in the growth medium for 10 min at 37°C, washed with growth medium, and then immediately imaged.
Cholesterol depletion, sequestering, replenishment, and quantitation
To deplete cellular cholesterol, cells were pretreated with 5 mmol/L methyl-β-cyclodextrin (MβCD; Sigma) for 30 min at 37°C and subjected to invadopodia assay in the presence of 1 mmol/L MβCD or water-soluble cholesterol balanced with MβCD (137 μmol/L cholesterol/1 mmol/L MβCD; Sigma) for cholesterol replenishment. To sequester membrane cholesterol, cells were pretreated with 50 μg/mL nystatin (Sigma) for 30 min at 37°C and tested for invadopodia activity in the continuous presence of nystatin. The relative amount of cellular cholesterol was determined with cholesterol/cholesterol ester quantitation kit (Calbiochem).
Cells were plated onto 10 cm gelatin-coated culture dishes at 1 × 106 per dish, and the cytosolic fraction (1 mL) was extracted with the ProteoExtract Subcellular Proteome Extraction kit (Calbiochem). Triton X-100 soluble materials were extracted with 500 μL TNE buffer [25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, and protease inhibitors] containing 1% Triton X-100. Insoluble materials were further extracted with 250 μL TNE buffer containing 1% SDS. Equal amounts of protein from each fraction were analyzed by immunoblotting.
Further details for all of these methods are available upon request.
Lipid rafts are enriched, internalized, and trafficked at invadopodia in human breast cancer cells
We first confirmed the localization of lipid rafts at invadopodia by using a lipid raft marker, CTxB; CTxB binds to lipid raft-enriched GM1 ganglioside and has been widely exploited to visualize lipid rafts (26). MDA-MB-231 invasive human breast cancer cells were plated onto a fluorescently labeled gelatin matrix and the lipid rafts of the plasma membrane were labeled with fluorescent CTxB. At the ventral surface of the cell, strong CTxB signals were observed at invadopodia, which were defined by the dot-like accumulation of F-actin associated with the degradation sites of the gelatin matrix (Fig. 1A). The green fluorescent protein (GFP)–tagged NH2-terminal sequence of Lyn (GFP-LynN), which was shown to target lipid rafts (27), was also accumulated at the gelatin degradation sites (Supplementary Fig. S1). Nevertheless, the GFP-LynN signals were more broadly distributed throughout the plasma membrane than CTxB signals, probably because this probe is less specific than CTxB.
To follow the course of lipid raft membranes at invadopodia, MDA-MB-231 cells were first labeled with Alexa 555-CTxB and cultured for 10 min to allow them to internalize lipid raft membranes. CTxB remaining on the cell surface was then stained with anti-CTxB antibody before cell permeabilization. Most of the signals for total and surface CTxB were colocalized on the plasma membrane (Fig. 1B). Internalized CTxB was then visualized by subtracting the surface CTxB signals from the total CTxB image. The resulting image showed that the signals for internalized CTxB are localized at F-actin–rich invadopodia (Fig. 1B). Time-lapse fluorescent microscopy was also carried out with MDA-MB-231 cells that stably expressed GFP-actin. The cells were labeled with Alexa 555-CTxB for 10 min and immediately imaged. CTxB signals were observed as small vesicles and some of them were localized around invadopodia (Fig. 1C; Supplementary Video 1). These vesicles were dynamically transported to and from invadopodia and actively fragmented into small vesicles or fused into large clusters (Fig. 1C). Taken together, these results indicate that invadopodia are enriched with lipid raft membranes where active raft membrane internalization and transport occur.
Lipid raft formation is necessary for invadopodia-mediated ECM degradation
The role of lipid raft formation in invadopodia-mediated gelatin degradation was then assessed. MDA-MB-231 cells were pretreated with MβCD, which depletes cellular cholesterol and therefore disassembles lipid rafts (28); the cells were then plated onto a gelatin matrix and tested for invadopodia formation. MβCD treatment inhibited invadopodia formation and degradation of the gelatin matrix (Fig. 2A and B). CTxB staining of MβCD-treated cells showed that patched signals of CTxB at the ventral membrane were decreased in these cells (Supplementary Fig. S2). The invadopodia formation and matrix degradation activity of MβCD-treated cells were restored by cholesterol replenishment (Fig. 2A and B). In this experimental condition, the amounts of cellular cholesterol in cells treated with MβCD and replenished with cholesterol were 41% and 175% of that of untreated cells, respectively (Supplementary Fig. S3A).
We also used the more specific lipid raft-inhibiting reagent nystatin, which binds and sequesters membrane cholesterol (29). Treatment of cells with 50 μg/mL nystatin significantly suppressed invadopodia-mediated gelatin degradation (Fig. 2C and D). At this concentration, nystatin had no significant effect on cell viability during the assay period (data not shown). Nystatin treatment had little effect on the amount of cellular cholesterol (Supplementary Fig. S3B), consistent with its cholesterol-sequestering but not extracting effect. These observations indicate that lipid raft formation is necessary for the assembly and functioning of invadopodia.
Cav-1 is an essential regulator of invadopodia formation
Caveolin is an essential component of caveolae, which are subtypes of lipid rafts, and is known to regulate the organization and dynamics of lipid rafts (17, 30). Therefore, the role of caveolin in invadopodia formation was investigated. MDA-MB-231 cells were transfected with siRNA targeting Cav-1 or Cav-2 and then tested for invadopodia formation. Immunoblot analysis confirmed the selective knockdown of the expression of each form of caveolin (Fig. 3A). Cells with a reduced amount of Cav-1 showed a significant decrease in invadopodia formation and gelatin degradation activity (Fig. 3B and C). Similar results were obtained with another siRNA targeting a different region of the Cav-1 gene (Supplementary Fig. S4A and B). The Cav-1 knockdown phenotype was rescued by retroviral reintroduction of mouse Cav-1, which has mismatches in the siRNA targeting sequence (Fig. 3A-C). In contrast, the knockdown of Cav-2 or Flot-1, a lipid raft-associated protein involved in endocytosis (31), had no significant effect on invadopodia formation (Fig. 3A and B). Although it was reported that knockdown of Flot-1 leads to downregulation of Cav-1 via lysosomal degradation (32), we could not detect any change in the amount of Cav-1 in Flot-1 knockdown cells in this experimental condition (Fig. 3A). It was recently reported that invadopodia formation in human melanoma cells is regulated by Cav-1–mediated control of membrane cholesterol levels (33). However, Cav-1 knockdown caused little change in the amount of cellular cholesterol, and the invadopodia formation in Cav-1 knockdown cells could not be rescued with provision of cholesterol in this cell type (Fig. 3D; Supplementary Fig. S4C). Similar results were obtained when lower concentrations of cholesterol were used for the provision (data not shown). These results suggest that Cav-1 is specifically involved in invadopodia formation by breast cancer cells independently of its role in the regulation of membrane cholesterol levels.
Next, the localization of endogenous Cav-1 at invadopodia was examined using immunocytochemistry. Cav-1 signals were detected as patched clusters distributed throughout the plasma membrane and vesicle-like pattern at the submembrane region (Fig. 4A). Accumulation of the signals was observed at invadopodia-mediated degradation sites of the gelatin matrix (Fig. 4A). Localization of Cav-1 was observed at some but not all invadopodia, suggesting that Cav-1 is not a structural component of invadopodia but rather has modifying functions required for proper invadopodia formation. Cav-1 was also colocalized with the internalized CTxB signals existing around invadopodia at the submembrane region (Fig. 4B). Thus, Cav-1 may be involved in the trafficking of lipid raft membranes at invadopodia.
Cav-1 regulates MT1-MMP–dependent matrix degradation activity
The invadopodia component that is coenriched with Cav-1 at lipid rafts was then determined. MDA-MB-231 cells cultured on gelatin-coated dishes were separated into cytosolic, Triton X-100 soluble, and Triton X-100 insoluble fractions. Lipid rafts are known enriched in the Triton X-100 insoluble fraction (34). The presence of major invadopodia components, including MT1-MMP, N-WASP, Arf6, cortactin, Cdc42, and Src, in each fraction was examined using immunoblotting (Fig. 5A). Among the invadopodia components examined, MT1-MMP was primarily detected in the Triton X-100 insoluble fraction, which is enriched with lipid raft proteins, Cav-1 and Flot-1.
Next, the localization of Cav-1 and MT1-MMP in association with invadopodia was examined. The mCherry-Cav-1 and GFP-MT1-MMP constructs were cotransfected into cells and the localization of these proteins was examined. Strong Cav-1 signals were observed as small vesicles concentrated around invadopodia and were colocalized with MT1-MMP signals (Fig. 5B). When analyzed using live-cell imaging, these small vesicles showed highly dynamic behavior, and CFP-Cav-1 and mCherry-MT1-MMP were cotrafficking with them in the cells (Fig. 5C; Supplementary Video 2).
To examine the involvement of Cav-1 in MT1-MMP function, MDA-MB-231 cells stably expressing mCherry-MT1-MMP were generated by retroviral transfection. When these cells were cultured on a fluorescent gelatin matrix, extensive gelatin degradation was detected throughout the cell body within 2 h (Fig. 5D; Supplementary Fig. S5A). At this time point, little gelatin degradation activity was detected in the parental MDA-MB-231 cells (Fig. 5D). Cav-1 knockdown significantly reduced the gelatin degradation activity of MT1-MMP–expressing cells (Fig. 5D). Interestingly, cholesterol depletion by MβCD treatment did not significantly affect the gelatin degradation by MT1-MMP cells, although this treatment efficiently reduced the amount of cellular cholesterol (Supplementary Fig. S5B and C). This observation implies that overexpression of MT1-MMP can overcome the effect of raft disruption. Altogether, these results indicate that Cav-1 regulates proper functioning of MT1-MMP.
Cav-1 expression is correlated with the invadopodia activity in human breast cancer cell lines
To further confirm that Cav-1 is an essential regulator of invadopodia formation, the correlation between Cav-1 expression and invadopodia activity in human breast cancer cell lines with different invasive phenotypes was determined. Cell lines tested were tumorigenic but noninvasive breast cancer cell lines T47D, SK-BR-3, and MDA-MB-453 and invasive breast cancer cell lines, Hs578T, BT549, and MDA-MB-231. When cultured on fluorescent gelatin-coated coverslips, the noninvasive cells showed only minimal formation of invadopodia and degradation of the gelatin matrix (Fig. 6A). In contrast, the invasive BT549, Hs578T, and MDA-MB-231 cells showed invadopodia formation associated with gelatin degradation (Fig. 6A). The expression of Cav-1 was readily detected only in cells possessing invadopodia activity (Fig. 6B). In contrast, no such correlation was observed in Flot-1 (Fig. 6B). The presence of processed active form of MT1-MMP (∼60 kDa) was also detected only in invasive cell lines and is enriched in Triton X-100 insoluble lipid raft fractions (Fig. 6C). On the other hand, inactive pro-form of MT1-MMP (∼63 kDa) was detected in all cell lines regardless of their invasive capacity (Fig. 6C). Finally, Cav-1 knockdown by siRNA significantly suppressed the invadopodia-mediated gelatin degradation activity of Hs578T and BT549 cells (Fig. 6D).
This study showed that lipid raft membranes are enriched, internalized, and actively trafficked at invadopodia and the disruption of lipid rafts suppresses invadopodia formation by human breast cancer cells. We reported previously that local actin polymerization mediated by N-WASP and the Arp2/3 complex is required for the assembly of invadopodia core structures (23). Two related reports showed that lipid rafts are the platforms for localized actin polymerization through the WASP/N-WASP-Arp2/3 complex pathway (35, 36). Therefore, lipid rafts may function in the recruitment of these actin nucleators, and possibly other invadopodia components, to the sites of invadopodia assembly. Because several invadopodia components, such as dynamin-2 and Arf6, are involved in raft endocytosis and trafficking (12, 37), these proteins may mediate the dynamic behavior of lipid rafts at invadopodia.
It has been shown that MT1-MMP resides in lipid rafts (38, 39) and increased localization of MT1-MMP in lipid rafts enhances invasion of prostate cancer cells (40). Consistent with these reports, the present study found that MT1-MMP is preferentially localized in lipid raft fractions among major invadopodia components. We observed that lipid raft disruption blocked the gelatin degradation activity of parental MDA-MB-231 cells, whereas it had little effect on that of the cells overexpressing MT1-MMP. This observation raises the possibility that endogenous MT1-MMP must be concentrated at invadopodia by lipid rafts to mediate focal matrix degradation, whereas overexpressed MT1-MMP can exert its activity without lipid raft-mediated concentration. This idea is supported by the fact that the cells overexpressing MT1-MMP degrade the gelatin matrix throughout the cell body irrespective of the presence of invadopodia.
In the present study, we showed that Cav-1 accumulates at invadopodia and its knockdown inhibits invadopodia formation. It was also shown that Cav-1 is expressed in invasive human breast cancer cell lines that possess an ability to form invadopodia. Supporting our observation, Cav-1 knockdown was reported to inhibit invadopodia formation in human melanoma cells (33). In breast cancer, Cav-1 is expressed in invasive and metastatic tumors and associated with poor patient prognosis (41–43). Furthermore, Joshi and colleagues recently reported that Cav-1 regulates invasion of human breast cancer cells (22). Therefore, our findings may provide a novel insight into the molecular mechanisms by which Cav-1 regulates the ECM remodeling associated with breast cancer cell invasion.
Several studies have reported that MT1-MMP is internalized, trafficked, and recycled to the cell surface through the caveolin-dependent pathway (38, 39). Additionally, Cav-1 has been shown to associate with MT1-MMP (44). We showed, for the first time, that MT1-MMP–containing vesicles are cotrafficked with Cav-1. Moreover, Cav-1 was coexpressed with active MT1-MMP in invasive breast cancer cell lines. These results suggest that Cav-1 regulates transport of lipid raft-associated MT1-MMP for functioning of invadopodia. We also found that Cav-1 knockdown, but not MβCD treatment, affects gelatin degradation activity of the cells overexpressing MT1-MMP. Consistent with this observation, it was reported that MβCD treatment does not disrupt the association of Cav-1 with MT1-MMP (44). As MβCD preferentially depletes cholesterol on the plasma membrane (45), it is possible that the regulation of MT1-MMP by Cav-1 occurs primarily in the intracellular pool of lipid rafts. Recently, two studies showed that MT1-MMP is delivered to invadopodia via targeted exocytosis by the exocyst complex and via targeted transport regulated by VAMP7 (46). As both the exocyst complex and VAMP7 are associated with lipid rafts (47, 48), Cav-1 and these molecules may coordinately regulate the MT1-MMP transport in the same lipid raft compartment. Contradicting our conclusion, it was reported that overexpression of Cav-1 inhibits the activity of coexpressed MT1-MMP (49). Because overexpression of Cav-1 compromises lipid raft-mediated endocytic pathway (50), the strict control of the expression level of caveoin-1 may be required for the proper trafficking of lipid raft-associated MT1-MMP.
Caldieri and colleagues reported that Cav-1 regulates invadopodia formation by modulating the balance of membrane cholesterol levels (33). In this study, we showed that the inhibition of invadopodia formation by Cav-1 knockdown could not be canceled by addition of cholesterol in breast cancer cells. Additionally, Cav-1 knockdown, but not MβCD treatment, blocked MT1-MMP–dependent gelatin degradation activity. These results indicate that Cav-1 regulates the MT1-MMP function and invadopodia formation independently of the balance of membrane cholesterol. Therefore, the present results imply that Cav-1 plays multiple roles in the trafficking of invadopodia components and that the roles of Cav-1 in cellular invasion differ to some extent among cancer cell types.
In conclusion, the results of this study provide evidence that lipid raft formation and Cav-1 are necessary for invadopodia functioning in human breast cancer cells. These findings provide new insight into the molecular mechanisms of invadopodia formation, which will contribute to the development of new therapeutic treatments for cancer invasion and metastasis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Grant-in-Aid for Scientific Research (B) and for Young Scientists (B), The Uehara Memorial Foundation, and The Kao Foundation for Arts and Sciences.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Toshio Kitamura for providing the Plat-A cells and pMXs vectors and Masahiro Kawamura, Yumiko Konko, Keiko Takayama, and Emi Muroi for technical assistance.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received June 26, 2009.
- Revision received August 24, 2009.
- Accepted September 9, 2009.
- ©2009 American Association for Cancer Research.