The Met receptor and alpha 6 beta 4 integrin can function independently to promote carcinoma invasion.

It has been proposed that a constitutive, physical association of the Met receptor and the alpha(6)beta(4) integrin exists on the surface of invasive carcinoma cells and that hepatocyte growth factor (HGF)-mediated invasion is dependent on alpha(6)beta(4) (Trusolino, L., Bertotti, A., and Comoglio, P. M. (2001) Cell 107, 643-654). The potential significance of these results prompted us to re-examine this hypothesis. Using three different carcinoma cell lines that express both Met and alpha(6)beta(4), we were unable to detect the constitutive association of these receptors by co-immunoprecipitation. Moreover, carcinoma cells that lacked expression of alpha(6)beta(4) exhibited Met-dependent invasion toward HGF, and increasing Met expression by viral infection of these cells enhanced invasion without inducing alpha(6)beta(4) expression. Although expression of alpha(6)beta(4) in such cells enhanced their invasion to HGF, it also enhanced their ability to invade toward other chemoattractants such as lysophosphatidic acid, and this latter invasion was not inhibited by a function-blocking Met antibody. Finally, depletion of beta(4) by RNA interference in invasive carcinoma cells that express both receptors reduced the ability of these cells to invade toward HGF by approximately 25%, but it did not abrogate their invasion. These data argue that the invasive function of Met can be independent of alpha(6)beta(4) and that alpha(6)beta(4) has a generic influence on the invasion of carcinoma cells that is not specific to Met.

It has been proposed that a constitutive, physical association of the Met receptor and the ␣ 6 ␤ 4 integrin exists on the surface of invasive carcinoma cells and that hepatocyte growth factor (HGF)-mediated invasion is dependent on ␣ 6 ␤ 4 (Trusolino, L., Bertotti, A., and Comoglio, P. M. (2001) Cell 107, 643-654). The potential significance of these results prompted us to re-examine this hypothesis. Using three different carcinoma cell lines that express both Met and ␣ 6 ␤ 4 , we were unable to detect the constitutive association of these receptors by co-immunoprecipitation. Moreover, carcinoma cells that lacked expression of ␣ 6 ␤ 4 exhibited Met-dependent invasion toward HGF, and increasing Met expression by viral infection of these cells enhanced invasion without inducing ␣ 6 ␤ 4 expression. Although expression of ␣ 6 ␤ 4 in such cells enhanced their invasion to HGF, it also enhanced their ability to invade toward other chemoattractants such as lysophosphatidic acid, and this latter invasion was not inhibited by a function-blocking Met antibody. Finally, depletion of ␤ 4 by RNA interference in invasive carcinoma cells that express both receptors reduced the ability of these cells to invade toward HGF by ϳ25%, but it did not abrogate their invasion. These data argue that the invasive function of Met can be independent of ␣ 6 ␤ 4 and that ␣ 6 ␤ 4 has a generic influence on the invasion of carcinoma cells that is not specific to Met.
Understanding the receptor-mediated mechanisms that underlie invasive carcinoma is a timely and significant endeavor. The involvement of specific integrins and growth factor receptors in the invasive process is established, and several lines of evidence indicate that these two classes of surface receptors may cooperate to effect a wide range of biological functions, including the migration and invasion of tumor cells (2)(3)(4). The available data indicate that integrin and growth factor signaling can be synergistic, and in some cases physical association may occur between these receptor types. Insight into the nature of such receptor interactions has important implications not only for understanding the biology of tumor invasion but also for the design and use of therapeutics targeted to these receptors (5).
An integrin of particular relevance to invasive carcinoma is ␣ 6 ␤ 4 (6 -8). This integrin, which is expressed primarily on the basal surface of most epithelia and in most carcinoma cells, is defined as an adhesion receptor for most of the known basement membrane laminins (6,9). A primary function of ␣ 6 ␤ 4 , revealed by studies of knock-out mice, is to maintain the integrity of epithelia (10,11). This critical role for ␣ 6 ␤ 4 derives from its ability to mediate the formation of stable adhesive structures, termed hemidesmosomes, on the basal cell surface that link the cytokeratin network with laminins in the basement membrane (12). Recent studies have revealed novel and important functions for this integrin in the migration and invasion of carcinoma cells (13). The expression of ␣ 6 ␤ 4 is maintained or often increased in invasive and metastatic carcinomas, and its expression level correlates with the progression of these carcinomas (14). More recently, compelling data were reported that suggest the ␣ 6 ␤ 4 integrin is essential for squamous carcinogenesis (15).
Given the potential importance of the ␣ 6 ␤ 4 integrin to invasive carcinoma, extensive efforts are being made to define the mechanisms by which it facilitates the invasive process. Advances include the observation that ␣ 6 ␤ 4 is localized to the leading edge of migrating carcinoma cells where it can contribute to the formation and stabilization of actin protrusions (16,17). In addition, there is evidence from several laboratories indicating that ␣ 6 ␤ 4 stimulates the activity of phosphoinositide 3-OH kinase (PI3K) 1 in invasive carcinoma cells and that PI3K is essential for migration and invasion (7,18). Interestingly, it has been suggested that ␣ 6 ␤ 4 activates PI3K and mediates invasion through its ability to cooperate with specific growth factor receptors (1,4,18). For example, ␣ 6 ␤ 4 has been shown to associate with erbB2 on the surface of breast carcinoma cells, and this interaction appears to facilitate activation of PI3K and invasion (18,19).
More recently, it was argued that ␣ 6 ␤ 4 functions as an essential adaptor protein for the Met receptor in invasive carcinoma cells (1). The impact of this finding is amplified by the fact that substantial evidence exists for the importance of Met in the scattering, invasion, and metastasis of tumor cells (20,21). If ␣ 6 ␤ 4 were an essential, specific adaptor for Met function in these events, the consequences for carcinoma biology and therapy would be profound. The potential significance of these results prompted us to re-examine the central findings of this study, which were that a selective physical association between Met and ␣ 6 ␤ 4 exists on the surface of invasive carcinoma cells and that Met cannot promote invasion in the absence of ␣ 6 ␤ 4 expression.

EXPERIMENTAL PROCEDURES
Cells-MDA-MB-231 and MDA-MB-435 breast carcinoma cells were obtained from the Lombardi Breast Cancer Depository at Georgetown University (Washington, DC), and A431 cells were purchased from American Type Culture Collection. Cells were grown in low glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 25 mM Hepes. The generation of stable transfectants of MDA-MB-435 cells that express the ␣ 6 ␤ 4 integrin has been described previously (7). For Met expression studies, a vesicular stomatitis virus-coated retrovirus containing Met cDNA was obtained from Dr. Morag Park (McGill University, Montreal, Quebec, Canada). SUM-159 cells were obtained from Dr. Stephen Ethier at the University of Michigan Comprehensive Cancer Center (Ann Arbor, MI) and maintained in Ham's F-12 medium containing 5% fetal bovine serum, 5 g/ml insulin, 1 mg/ml hydrocortisol, 1% penicillin-streptomycin, and 25 mM Hepes (22).
To generate retroviruses, ␤ 4 siRNA or ␤ 4 SCR-pSUPER.retro and expression plasmids containing envelope and packaging proteins required for viral propagation (Imgenex, San Diego, CA) were transfected into 293T cells (3 ϫ 10 6 cells/100-mm plate) with LipofectAMINE (Invitrogen) as described by the manufacturer. Three days after transfection, viral supernatants were harvested, and SUM-159 recipient cells were infected in the presence of a serum-containing medium supplemented with 8 g/ml polybrene (Sigma). Following infection for 24 h, resistant cells were selected with 4.0 g/ml puromycin (Clontech), and cell lines that stably expressed ␤ 4 siRNA or ␤ 4 SCR were generated. The stable ␤ 4 siRNA SUM-159 cell line was then sorted by flow cytometry, and the population of cells that expressed the lowest level of ␤ 4 was isolated. The sorting and subsequent surface labeling analysis of the ␤ 4 -deficient cell line was performed with 3E1, a mouse anti-␤ 4 integrin antibody (Chemicon, Temecula, CA). The decreased level of ␤ 4 expression in this cell line was further confirmed by Western blotting with a rabbit polyclonal anti-␤ 4 integrin antibody (505) (17).
Biochemical Analyses-For the co-immunoprecipitation studies, cells were extracted for 15 min at 4°C with a buffer containing 50 mM Hepes (pH 7.4), 5 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, and 1% Nonidet P-40 in the presence of protease and phosphatase inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 mg/ml each of aprotinin, leupeptin, pepstatin, sodium fluoride, and sodium orthovanadate). Extracts were clarified by centrifugation at 13,000 rpm for 10 min, the supernatants were collected, and their protein concentrations were determined using the Bio-Rad DC protein assay kit. Immunoprecipitations were performed with equal amounts of total protein. Extracts were preabsorbed overnight using protein A-and G-Sepharose beads (Amersham Biosciences) to prevent nonspecific binding. After centrifugation at 2000 rpm for 5 min to pellet these beads, the supernatants were incubated overnight at 4°C with 1 g of the following antibodies: rat ␤ 4 mAb, 439-9B (obtained from Rita Falcioni, Regina Elena Cancer Institute, Rome, Italy), polyclonal anti-human Met C-12 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse ␤ 4 mAb, 3E1 (Chemicon), mouse Met mAb DO-24 (Upstate Biotechnology, Lake Placid, NY), and the appropriate rat, rabbit, and mouse IgGs (Sigma). Immune complexes were precipitated with protein A-and G-Sepharose, washed four times with extraction buffer, and eluted in 1ϫ reducing sample buffer.
For immunoblotting, cell extracts were prepared as described previously. These extracts or the immune complexes were separated by SDS-PAGE and transferred to Hybond-C nitrocellulose membranes (Amersham Bioscience). Membranes were blocked for 1 h using 50 mM Tris buffer (pH 7.5) containing 0.15 M NaCl, 0.05% Tween 20 (TBS-T), and 5% Carnation dry milk. The membranes were incubated overnight in the same buffer containing antibodies specific for either the ␤ 4 integrin (505), anti-␤-actin (Sigma), or Met (C-12, Santa Cruz Biotech-nology, or (DO-21, Upstate Biotechnology). After three 10-min washes in TBS-T, the membranes were incubated for 1 h in blocking buffer containing horseradish peroxidase-conjugated secondary antibodies. After three 10-min washes in TBS-T, proteins were detected by enhanced chemiluminescence (Pierce).
For immunoblots involving the phosphotyrosine Ab (anti-phosphotyrosine, clone PY99, Cell Signaling Technology, Beverly, MA), the membranes were blocked for 1 h using 10 mM Tris buffer (pH 7.5) containing 0.5 M NaCl, 0.1% Tween 20, and 2% bovine serum albumin (w/v). The membranes were washed briefly and then incubated overnight at 4°C in the same blocking buffer containing the phosphotyrosine Ab. After washing, the filters were incubated for 1 h in blocking buffer containing horseradish peroxidase-conjugated secondary Ab, and the proteins were detected by enhanced chemiluminescence.
Invasion Assays-To prepare the Transwell membranes (Corning Glass) for the invasion assays, 0.5 g of Matrigel (Collaborative Research, Waltham, MA) was diluted with cold water and left to dry overnight onto the upper side of the membranes at 25°C. On the following day, the lower sides of the membranes were coated with 10 g/ml laminin-1 (Calbiochem) and again left to dry overnight at 4°C. The membranes were blocked with Dulbecco's modified Eagle's medium (or Ham's F-12 medium for SUM-159) for 1 h at 37°C. Cells were trypsinized on the following day and resuspended in Dulbecco's modified Eagle's medium or Ham's F-12 medium containing 0.25% heatinactivated lipid-free bovine serum albumin, and a total of 5 ϫ 10 4 cells was added to the upper chamber of each well. Chemoattractants (50 ng/ml HGF (R & D System, Minneapolis, MN), 100 ng/ml lysophosphatidic acid (Sigma), or 3T3 cell-conditioned medium) were added to the bottom wells in Dulbecco's modified Eagle's medium-bovine serum albumin. In some assays, cells were preincubated with a function-blocking Met mAb, clone 95309 (R & D Systems), for 1 h at 4°C before plating. After incubating for 2 or 4 h at 37°C, non-migrating cells were mechanically removed from the upper chamber with a cotton swab. Cells that had migrated to the lower side of the Transwell membrane were fixed with 100% methanol and stained with 0.2% crystal violet in 2% ethanol. Invasion was quantified by counting the number of cells per square millimeter using a reticule with bright-field optics.

Lack of Evidence for Constitutive Association between Met and ␣ 6 ␤ 4 Integrin in Invasive Carcinoma
Cells-Evidence to support a physical association between Met and the ␣ 6 ␤ 4 integrin has been shown in co-immunoprecipitation experiments using COS cells transfected to express high levels of both Met and ␣ 6 ␤ 4 , as well as in carcinoma cells that express both receptors endogenously (1). In particular, it was reported that ␤ 4 immunoprecipitates of A431 cells contain significant amounts of Met. To confirm these findings, we performed co-immunoprecipitation assays using A431 cells, as well as MDA-MB-231 breast carcinoma cells, which express high levels of both ␣ 6 ␤ 4 and Met. In addition, MDA-MB-435 cells that stably express the ␣ 6 ␤ 4 integrin were examined because they had been used previously to demonstrate the necessity of ␣ 6 ␤ 4 for HGF-dependent invasion (1). To assess a putative physical association of these two receptors, cells were extracted with the same Nonidet P-40 (1%) buffer used previously (1), and extracts were "precleared" and then immunoprecipitated with Abs specific for either the ␤ 4 integrin subunit (439-9B) or Met (C-12). Subsequently, the precipitates were analyzed by immunoblotting with either ␤ 4 -specific polyclonal (505) Ab or Met Ab (C-12). As shown in Fig. 1A, the ␤ 4 integrin subunit was evident in the ␤ 4 immunoprecipitates, and Met was evident in the Met immunoprecipitates. However, there was no indication of Met in the ␤ 4 immunoprecipitates or of ␤ 4 in the Met immunoprecipitates. To exclude the possibility that the co-immunoprecipitation of these two receptors is dependent on the detergent used for extraction, we also used Triton X-100-based buffer (1%) but failed to detect evidence for their physical association (data not shown). In addition, other Abs were used for the co-immunoprecipitation studies in A431 cells. Two different Met Abs (C-12 and DO-24) immunoprecipitated Met effectively but not the ␤ 4 integrin subunit (Fig. 1B). Similarly, ␤ 4 integrin subunit Abs (439-9B and 3E1) immunoprecipitated the ␤ 4 integrin subunit but not Met (Fig. 1B).
Met Can Function Independently of the ␣ 6 ␤ 4 Integrin in MDA-MB-435 Cells-To evaluate the hypothesis that the invasive function of Met is dependent on the ␣ 6 ␤ 4 integrin, we used MDA-MB-435 cells that express Met but not ␣ 6 ␤ 4 (Fig. 2). In addition, the level of Met expression was increased in these cells by retroviral infection with a Met cDNA. This infection increased Met expression by ϳ2-fold ( Fig. 2A), but it did not induce expression of ␣ 6 ␤ 4 (Fig. 2B). The ability of Met to signal in the absence of ␣ 6 ␤ 4 expression was examined. In the control cells, HGF stimulation induced a rapid and marked increase in tyrosine phosphorylation of Met as assessed by immunoprecipitating Met followed by immunblotting these immunoprecipitates with a phosphotyrosine-specific Ab (Fig. 2C). The intensity of this phosphotyrosine signal was increased in the Met infectants, in agreement with the fact that these cells express significantly more Met than do the control infectants ( Fig. 2A).
Subsequently, we assessed the ability of the control and Met infectants to invade toward HGF. Invasion assays were performed for 4 h without prior serum deprivation. As shown in Fig. 2D, HGF induced the invasion of the control cells significantly, and this invasion was abrogated by a function-blocking Met-specific antibody. A 2-fold induction of HGF-dependent invasion was observed in the Met infectants compared with control cells, substantiating the conclusion that Met promotes invasion in the absence of ␣ 6 ␤ 4 integrin (Fig. 2D). ␣ 6 ␤ 4 Integrin Has a Generic Influence on Carcinoma Invasion That Is Not Specific to Met-A key finding in the previous study, which concluded that ␣ 6 ␤ 4 is necessary for the invasive function of Met, was that expression of ␣ 6 ␤ 4 in MDA-MB-435 cells induces their ability to invade toward HGF (1). Although our results confirm the observation that expression of the ␣ 6 ␤ 4 integrin increases the invasion of these cells toward HGF, we observed that the mock transfectants, which lack ␣ 6 ␤ 4 , exhibited significant migration toward HGF (Fig. 3A). Moreover, the level of invasion induced by expression of ␣ 6 ␤ 4 is comparable with the increased invasion that results from increased Met expression and that this latter mode of invasion occurs in the absence of ␣ 6 ␤ 4 (Fig. 2D). Furthermore, expression of ␣ 6 ␤ 4 in MDA-MB-435 cells also enhanced the ability of the MDA-MB-435 cells to invade toward other chemoattractants such as lysophosphatidic acid, and this invasion was not inhibited by a function-blocking Met antibody (Fig. 3B). This latter result indicated that the expression of ␣ 6 ␤ 4 can enhance the ability of MDA-MB-435 cells to invade independently of Met expression. Invasion assays were performed for 4 h in serum-free medium, and, under these conditions, no significant increase in apoptosis was observed for any cell population as assessed by annexin-V fluorescein isothiocyanate staining (data not shown).
To assess the putative functional dependence of Met on the ␣ 6 ␤ 4 integrin from a different perspective, we used SUM-159 cells (22). These invasive breast carcinoma cells express both ␣ 6 ␤ 4 and Met (Fig. 4A). Using this cell line, we generated a SUM-159 cell line deficient in ␤ 4 integrin expression using siRNA strategies (23). Stable infectants that exhibited a reduction in ␤ 4 expression were sorted by fluorescence-activated cell sorter using a ␤ 4 -specific antibody, and a population of cells was isolated that exhibited no detectable ␤ 4 expression as evident by immunoblotting (Fig. 4A). Notably, the loss of ␤ 4 expression had no effect on Met expression in these cells (Fig.  4A). SUM-159 cells exhibited a robust invasion toward HGF FIG. 1. Lack of evidence for a constitutive association between Met and the ␣ 6 ␤ 4 integrin. A, extracts from the indicated cell lines were immunoprecipitated (IP) with antibodies against Met (clone C-12), ␤ 4 integrin (clone 439-9B), or the indicated IgG control (rbIgG, rabbit IgG; rIgG, rat IgG). Immunoblot analysis of these immunoprecipitates was performed using anti-Met (clone C-12) and anti-␤ 4 integrin polyclonal (505) antibodies. B, extracts from A431 cells were immunoprecipitated with ␤ 4 integrin-specific antibodies (mouse monoclonal clone DO-24 and rabbit polyclonal clone C- 12) or IgG controls (mouse IgG and rat IgG), and immunoprecipitates were analyzed by immunoblotting as described in A. (Fig. 4B). Loss of ␣ 6 ␤ 4 expression reduced the ability of these cells to invade toward HGF by ϳ25%, but it did not abrogate their invasion (Fig. 4B). Moreover, loss of ␣ 6 ␤ 4 expression also diminished the invasion of SUM-159 cells toward 3T3 cellconditioned medium by ϳ30%.

DISCUSSION
The report that the invasive function of the Met receptor is dependent on a physical association with the ␣ 6 ␤ 4 integrin, which provides a "signaling adaptor function," afforded a compelling model for invasive carcinoma that linked these two receptors (1).
The data obtained in our study unfortunately do not support the central tenets of this model. Rather, our data argue that the invasive function of Met can be independent of ␣ 6 ␤ 4 and that ␣ 6 ␤ 4 has a generic influence on the invasion of carcinoma cells that is not specific for HGF-dependent invasion.
The demonstration of a physical association between an integrin and a growth factor receptor provides prima facie evidence for cooperativeness of function. For this reason, the previous finding that Met and ␣ 6 ␤ 4 could be co-immunoprecipitated from GTL-16 cells, which overexpress a constitutively active form of Met, from COS cells engineered to express both receptors at high levels and from A431 cells, which also express both receptors, strengthened the possibility of a functional dependence (1). Despite using three different carcinoma cell lines that express both Met and ␣ 6 ␤ 4 (A431, MDA-MB-231, and MDA-MB-435, engineered to express ␣ 6 ␤ 4 ), however, we were unable to detect any evidence for the constitutive association of these receptors by co-immunoprecipitation. The reason for the difference between our results and those of Trusolino et. al. (1) is unclear. It is worth noting, however, that in the previous study co-immunoprecipitation data were not provided for either MDA-MB-231 cells or MDA-MB-435/␤ 4 cells, and an association between Met and ␣ 6 ␤ 4 in A431 cells was detected only by immunoprecipitation with a ␤ 4 integrin antibody and immunoblotting with a Met Ab and not vice versa. In addition, the use of another purified ␤ 4 integrin Ab (439-9B) failed to coimmunoprecipitate Met. Although our data refute the existence of a constitutive association of Met with ␣ 6 ␤ 4 in carcinoma cells, they do not exclude the occurrence of a transient association between these two receptors in certain physiological situations or the possibility that a spurious association may occur on their gross overexpression.
The lack of evidence for a physical association between ␣ 6 ␤ 4 and Met does not negate the possibility that they exhibit functional cooperativeness. To evaluate the hypothesis that the invasive function of Met depends on ␣ 6 ␤ 4 , we assessed the invasion of MDA-MB-435 cells, which express Met but not ␣ 6 ␤ 4 . These cells exhibited significant invasion toward HGF, and their rate of invasion increased in response to increasing Met expression by retroviral infection. The fact that these cells are capable of significant HGF-dependent invasion in the absence of ␣ 6 ␤ 4 expression argues against the necessity of this integrin for Met function. Moreover, a key finding in the pre- vious study was that expression of ␣ 6 ␤ 4 in MDA-MB-435 cells induced their ability to invade toward HGF but not toward EGF. We note, however, EGF is not a suitable negative control because MDA-MB-435 cells lack expression of the EGF receptor (24). 2 Additional support for the hypothesis that the invasive function of Met can occur independently of ␣ 6 ␤ 4 is provided by our data on SUM-159 breast carcinoma cells. These invasive cells express both Met and ␣ 6 ␤ 4 , and they exhibit a robust invasion toward HGF. Our finding that elimination of ␣ 6 ␤ 4 expression using a ␤ 4 -specific siRNA reduced but did not abrogate invasion toward HGF, however, argues against the conclusion that ␣ 6 ␤ 4 is an essential adaptor for Met in promoting carcinoma invasion. The strength of the SUM-159 data, in contrast to MDA-MB-435 cells, is that this cell line exhibits endogenous expression of both receptors, and the assumption can made that if Met function were dependent on ␣ 6 ␤ 4 , it should be evident in such a cell line.
Based on several studies as well as the findings reported here, a consensus is emerging that ␣ 6 ␤ 4 cooperates with growth factor receptors to promote carcinoma invasion and other functions (7,18,25). Perhaps the most conclusive evidence in this regard is the finding that macrophage-stimulating protein on binding to its receptor, the Ron tyrosine kinase, promotes an association between Ron and ␣ 6 ␤ 4 that results in PI3K activation and consequent migration (4). There is also evidence that ␣ 6 ␤ 4 can cooperate with erbB2 in breast carcinoma cells to 2 R. Bachelder, personal communication.

FIG. 4. Loss of ␣ 6 ␤ 4 integrin expression in SUM-159 cells reduces but does not abrogate invasion toward HGF and 3T3 cellconditioned medium.
A, SUM-159 cells stably expressing either scrambled (scr) or ␤ 4 integrin RNAi (␤ 4 siRNA) oligonucleotides were extracted, and equal amounts of protein extracts were analyzed by immunoblot analysis using Met (C-12), ␤ 4 (505), and actin-specific Abs. B, the ability of the cells described in A to invade Matrigel toward HGF (50 ng/ml) or 3T3 cell-conditioned medium was investigated in a 2-h assay. The mean number of invasive cells (Ϯ S.D.) from the five independent field per well is indicated on the y axis. Similar data were obtained in three separate experiments. activate PI3K and promote invasion (18). The conclusion that the function of one specific growth factor receptor (Met) is absolutely dependent on ␣ 6 ␤ 4 for promoting invasion, however, is not supported by our data. A more appropriate assessment of the relationship between Met and ␣ 6 ␤ 4 would be that expression of ␣ 6 ␤ 4 can enhance invasion toward several growth factors, including HGF and lysophosphatidic acid, as well as those present in 3T3 cell-conditioned medium (Figs. 3 and 4). At the same time, our results indicate that the ability of Met to promote invasion is not dependent on ␣ 6 ␤ 4 in these cells and that increasing Met expression in the absence of ␣ 6 ␤ 4 enhances HGF-mediated invasion.
The mechanism that underlies the ability of ␣ 6 ␤ 4 to promote invasion likely involves its ability to stimulate PI3K. Compelling evidence exists for ␣ 6 ␤ 4 -mediated activation of this enzyme by mechanisms that include phosphorylation of insulin receptor substrate proteins (26), cooperation with erbB2 (18,19) and Ron (4), and the elaboration of vascular endothelial growth factor autocrine signaling (27). In addition, the regulated expression of specific transcription factors such as nuclear factor of activated T cells by ␣ 6 ␤ 4 may contribute to the invasive phenotype (28). Clearly, Met is one of several growth factor receptors with a function that may be enhanced by ␣ 6 ␤ 4 expression but that can signal and promote invasion in the absence of ␣ 6 ␤ 4 expression. The challenge ahead is to define the mechanisms by which expression of ␣ 6 ␤ 4 enhances the function of multiple growth factor receptors.