ErbB2 (HER2, Neu) and Ras play key roles in tumor invasion and metastasis. We identified a novel mechanism by which integrin α6β4 regulates ErbB2 expression, Ras activation, and the invasion of breast carcinoma cells. Here we show that integrin α6β4 regulates Ras activity especially in serum-depleted condition. Down-regulation of β4 integrin by β4 short hairpin RNA (shRNA) decreased Ras activity and carcinoma invasion whereas reexpression of this integrin restored Ras activity. ErbB2, a binding partner of epidermal growth factor receptor (EGFR), and EGFR modulated Ras activity, and integrin α6β4 regulated phospho-EGFR level without affecting EGFR expression. We also found that integrin α6β4 is involved in ErbB2 expression. Depletion of β4 by shRNA reduced ErbB2 protein level without affecting ErbB2 mRNA level and reexpression of β4 increased ErbB2 protein level. Reduction of eukaryotic initiation factor 4E, a rate-limiting factor for cap-dependent translation, decreased ErbB2 protein level, and β4 shRNA cells exhibited a shift in ErbB2 mRNA to light polysomes compared with control cells. These results show that integrin α6β4 regulates ErbB2 through translational control. In summary, we propose a novel mechanism for ErbB2 up-regulation and Ras activation in serum-depleted breast cancer cells; integrin α6β4 regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. These findings provide a mechanism that substantiates the reported role of α6β4 in carcinoma invasion. (Cancer Res 2006; 66(5): 2732-9)

Integrins regulate cell adhesion to the extracellular matrix and relay molecular cues about the cellular environment that influence cell shape, survival, proliferation, gene transcription, and migration (1). Integrins have short cytoplasmic domains but an exception is integrin α6β4. Integrin β4 has an atypical cytoplasmic domain; two pairs of fibronectin type III repeats separated by a connecting segment characterize this domain and it is distinct both in size (∼1,000 amino acids) and structure from any other integrin subunit. This integrin is often up-regulated in carcinoma cells and there is compelling evidence that α6β4 facilitates the formation of some carcinomas as well as the migration, invasion, and survival of carcinoma cells (24). Recently, α6β4 integrin was found to promote the onset of the invasive phase of pathologic angiogenesis (5).

Sustained activation of the Ras GTPase is observed in a significant fraction of invasive breast carcinomas and it has been linked to the development and progression of this disease (6). Interestingly, however, mutations in the Ras gene are relatively infrequent in breast cancer (<5% of tumors; ref. 7). These data have led to the hypothesis that persistent activation of signaling pathways upstream of Ras accounts for its activation in invasive breast carcinomas. In this direction, signaling through EGF family receptors is known to activate Ras in many cellular contexts. For breast cancer, in particular, ErbB2 (HER2, Neu), which is persistently activated in ∼30% of breast cancers, has been implicated in Ras activation (8). What has emerged from these findings is that the ErbB2/Ras pathway is a critical signaling pathway in breast cancer and a prime target for therapeutic intervention (9).

Many growth factor receptors have been shown to interact with integrins to transduce stronger and more efficient signals to downstream molecules (10). ErbB2 can associate with integrin α6β4 on the surface of breast carcinoma cell lines (11). Subsequent studies showed that both ErbB2 and integrin α6β4 are required for phosphoinositide 3-kinase (PI3K) activation and the stimulation of cell invasion (12). Despite the fact that ErbB2 is a major regulator for Ras activity and ErbB2 interacts with integrin α6β4, little is known about whether integrin α6β4 can regulate Ras activity via ErbB2-dependent pathway and how these three molecules relate to each other. Given the important roles of integrin α6β4, ErbB2, and Ras in breast carcinoma cells, we sought to investigate a more detailed relationship among α6β4, Ras, and EGF receptor signaling in these cells, especially those cells that exhibit persistent Ras activation. More specifically, we focused on cells that can sustain Ras activation in the absence of exogenous growth factor stimulation. The results obtained indicate that Ras activation in such cells requires expression of α6β4, which can phosphorylate the epidermal growth factor receptor (EGFR). Interestingly, further analysis revealed that α6β4 regulates eukaryotic initiation factor 4E (eIF4E) activity and translation of ErbB2.

Cells and reagents. SUM-159 breast carcinoma cells were obtained from Dr. Steve Ethier (University of Michigan, Ann Arbor, MI). Cells were maintained in Ham's F-12 supplemented with 5% fetal bovine serum, 5 μg/mL insulin, 1 μg/mL hydrocortisone, 100 units/mL penicillin, and 100 μg/mL streptomycin. β4 short hairpin RNA (shRNA)-pSuper.Retro and β4 Scr-pSuper.Retro vectors were generated and these were stably expressed in SUM-159 cells as previously described (13). The sorting and subsequent surface labeling analysis of the β4-deficient cell line were done with 3E1, a mouse anti-β4 integrin antibody (Chemicon, Temecula, CA). The decreased level of β4 expression in this cell line was further confirmed by Western blotting with a rabbit polyclonal anti-β4 integrin antibody (505). The β4 was reexpressed in the β4 shRNA cells with a human β4 retrovirus. The β4 retrovirus was generated by cotransfecting 293T cells with β4-pCLXSN (Alex Toker, Beth Israel Deaconess Medical Center, Boston, MA) and expression plasmids containing envelope and packaging proteins required for viral propagation using Lipofectamine as described by the manufacturer. Following infection, resistant cells were selected with 250 μg/mL G418 and β4 shRNA cells that reexpressed β4 were generated. Robust expression of β4 is permitted in these cells because the enzymatic complex responsible for shRNA is saturable at high expression levels. For cells expressing 5R (a c-erbB2-specific single-chain antibody; Nancy E. Hynes, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland), pBABE-5R and pBABE-control plasmids were introduced into the GP2-293 cells with vesicular stomatitis virus-G plasmids, and viral supernatants were used to infect SUM-159 cells. EGFR, phospho-EGFR, and phospho-tyrosine antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). ErbB2 and ErbB3 antibodies were purchased from NeoMarkers (Fremont, CA). A pan-ras antibody was purchased from Oncogene (Cambridge, MA). An eIF4E antibody was obtained from Transduction Laboratories (San Diego, CA). EGF was from Sigma (St. Louis, MO) and AG1478 was obtained from Calbiochem (San Diego, CA).

Apoptosis assays. Cells were washed once with serum-containing medium, once with PBS, once with Annexin V-FITC buffer [10 mmol/L HEPES-NaOH (pH 7.4), 140 mmol/L NaCl, 2.5 mmol/L CaCl2], and then incubated for 15 minutes at room temperature with 5 μg/mL Annexin V-FITC (Biosource International, Camarillo, CA). After washing once with Annexin V buffer, the samples were resuspended in the same buffer. Immediately before analysis, 5 μg/mL propidium iodide (Biosource International) was added to distinguish apoptotic cells from necrotic cells, and the cells were analyzed using a flow cytometer.

Transfections. The eIF4E-sense and eIF4E-antisense constructs were a kind gift from Dr. Arigo De Benedetti (Louisiana State University, LA). The Dominant-negative H-Ras (N17 H-Ras) was kindly provided by Dr. Kun-Liang Guan (University of Michigan, Ann Arbor, MI). Cells were transfected with LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer. The transfection efficiency for SUM-159 cells was 55% to 60% as determined by green fluorescent protein plasmid transfection.

Invasion assays. The lower and upper parts of Transwells (Corning Costar) were coated with 10 μL of type I collagen (0.5 mg/mL) and 0.5 μg of cold water–diluted Matrigel (Collaborative Research, Bedford, MA), respectively. Subsequently, the coated Transwell membranes were incubated with Ham's F-12 media for 1 hour and 5 × 104 cells were plated in the upper chambers. The Ham's F-12 in the lower chambers contained 0.1 mg/mL bovine serum albumin (BSA). The inserts were incubated for 8 hours for cells that were grown in the presence of serum or for 8 hours for cells that were grown in the serum-depleted media for 28 hours. The cells that had invaded the lower surface of the membrane were fixed with methanol and stained with 0.2% crystal violet in 2% ethanol. The number of cells that had invaded was quantified by counting random fields using a light microscope equipped with a reticle.

Ras assay. Ras activity was measured utilizing a glutathione S-transferase (GST) fusion protein containing the Ras-binding domain (RBD) of Raf-1 as described (14). GST-Raf1-RBD construct was kindly provided by Dr. Johannes L. Bos (University Medical Centre Utrecht, Netherlands). The plasmid was transformed into Escherichia coli strain and protein production was initiated by adding isopropyl-d-thiogalactopyranoside to the cultures. Bacteria were resuspended in sonication buffer [20% sucrose, 10% glycerol, 50 mmol/L Tris-HCl (pH 8.0), 2 mmol/L DTT, 2 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 2 μg/mL aprotinin] and lysed by sonication. The lysate was centrifuged at 12,000 × g for 1 hour in the cold. Bacterially produced GST-Raf1-RBD was precoupled to glutathione-agarose beads and washed in cell lysis buffer [1% NP40, 10% glycerol, 50 mmol/L Tris (pH 7.4), 200 mmol/L NaCl, 2.5 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L sodium orthovanadate, 1 μg/mL leupeptin, 2 μg/mL aprotinin, 10 μg/mL trypsin inhibitor, 10 μg/mL NaF]. The cells were lysed in cell lysis buffer and cleared lysates were split and used for the determination of Ras activity or for the analysis of protein expression. For Ras assay, cleared lysates were incubated with bead-GST mixture for 45 minutes. The beads mixtures were then washed four times with cell lysis buffer and bound proteins were eluted with SDS-PAGE sample buffer. Samples were separated by SDS-PAGE (12.5% polyacrylamide), blotted, and probed with anti-Ras antibody.

Analysis of protein expression. Cells were analyzed for their expression of specific proteins by either fluorescence-activated cell sorting (FACS) or immunoblotting. For FACS analysis, cells were washed twice with ice-cold PBS containing 0.2% BSA. Aliquots of cells were incubated for 1 hour at 4°C with antibodies in the PBS/BSA solution. The cells were washed thrice with PBS/BSA and then incubated with secondary antibodies coupled to R-phycoerythrin for 1 hour at 4°C. After washing thrice with PBS/BSA, the cells were resuspended in PBS and analyzed using a FACScan (Becton Dickinson, Franklin Lakes, NJ). Immunoblot analysis was done as previously described (15).

Immunoprecipitation. Cells were extracted with a buffer [20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mg/mL aprotinin, 2 mg/mL leupeptin, and 1 mg/mL pepstatin]. After centrifugation, supernatants were collected and these were preabsorbed for 2 hours with protein A-Sepharose and protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ). After centrifugation at 2,500 rpm for 5 minutes, the supernatants were incubated overnight at 4°C with ErbB2 antibody. After precipitation with protein A-Sepharose and protein G-Sepharose, these were washed four times with a buffer and eluted in 2× reducing sample buffer.

Polysome isolation. Cells were maintained in medium without serum for 36 hours and then pretreated with 100 μg/mL cycloheximide for 15 minutes at 37°C before being harvested. After washing once with PBS containing 100 μg/mL cycloheximide, the cells were resuspended in 0.5 mL of a modified U+S buffer (16). This buffer was composed of 200 mmol/L Tris-HCl (pH 8.8), 25 mmol/L MgCl2, 5 mmol/L EGTA (pH 8.0), 150 mmol/L KCl, 10 μg/mL heparin, 5 mmol/L DTT, 1% sodium deoxycholate, 2% polyoxyethylene 10-tridecy ester, 100 μg/mL cycloheximide, and 200 mmol/L sucrose. RNase inhibitor (Amersham Biosciences) was added to a final concentration of 0.5 units/μL. Cells were homogenized with 20 to 25 strokes in a Kontes tissue homogenizer, followed by centrifugation for 5 minutes at 14,000 × g. The supernatant was collected and frozen at −80°C until further use. Sucrose gradients (15-50%, w/w) were layered with 300 μL of cleared cell extract, which was then centrifuged at 160,000 × g for 2 hours. Fractions (0.75-0.375 mL) were withdrawn from the top of the gradient and monitored for absorbency at 254 nm using an Isco syringe pump with UV-6 detector. Total RNA from the sucrose gradient fractions was extracted using Trizol LS (Life Technologies, Rockville, MD) according to the instructions of the manufacturer. Northern blot analysis was used to measure the mRNA level in each fraction.

Northern blot analysis. RNAs were electrophoresed on 1% agarose gels containing 6% formaldehyde and transferred to Hybond-N membrane (Amersham Biosciences) by capillary transfer. The membrane was fixed using an optimized UV cross-linking procedure. Prehybridization and hybridization were done at 68°C in ExpressHyb hybridization solution (Clontech, Palo Alto, CA). cDNA probes were labeled with [32P]dCTP (3,000 Ci/mmol; Perkin-Elmer, Norwalk, CT) using a random primer kit (New England Biolabs, Ipswich, MA). The blot was washed twice with 20× SSC [300 mmol/L NaCl, 30 mmol/L sodium citrate (pH 7.0)] containing 0.05% SDS at 25°C and then with 0.1× SSC containing 0.1% SDS at 55°C and autoradiographed at −70°C.

Constitutive activation of Ras in serum-depleted cells requires expression of the α6β4 integrin. To examine the relationship between the α6β4 integrin and constitutive Ras activation, we used a retrovirus containing a β4 shRNA to deplete β4 expression in SUM-159 cells, which were derived from an aggressive, anaplastic breast tumor (17). This approach, which we previously described (13), resulted in the generation of a β4-RNAi cell line that exhibited a substantial reduction in total β4 expression as well as cell-surface β4 level compared with cells that expressed a scrambled shRNA sequence (Fig. 1A). To control for the specificity of the β4 shRNA in subsequent functional experiments, we reexpressed the human β4 integrin in the β4-RNAi cells to generate a β4-RNAi + β4 SUM-159 cell line that expresses the β4 integrin at a relatively high level (Fig. 1A).

SUM-159 cells exhibit constitutive Ras activation that is not dependent on the presence of serum (Fig. 1B). Interestingly, however, the ability of these cells to sustain Ras activation in serum-deprived cells depends on expression of α6β4 (Fig. 1B). Depletion of β4 expression had no effect on Ras activity in cells maintained in the presence of growth factor–rich serum. The specificity of this effect is evidenced by the fact that enhanced expression of β4 in the β4 shRNA cells significantly increased Ras activity in comparison with control cells (Fig. 1B).

A functional consequence of the influence of integrin α6β4 expression on Ras activity is invasion. As shown in Fig. 2A, depletion of β4 integrin expression resulted in a significant decrease in the ability of serum-depleted SUM-159 cells to invade Matrigel (Fig. 2A), a process that was also impeded by expression of a dominant-interfering Ras construct (Fig. 2B). Of note, loss of β4 integrin expression did not increase the apoptotic rate under these conditions (Fig. 2C).

Constitutive Ras activation depends on EGFR and ErbB2. Given the extensive literature on Ras activation by EGF family receptors and the importance of these receptors in breast cancer (18), we investigated the contribution of EGFR and ErbB2 to Ras activation in SUM-159 cells. These cells lack expression of ErbB3 (data not shown). Exposure of cells to AG1478, a specific inhibitor of the EGFR, resulted in a concentration-dependent reduction in Ras activity in serum-deprived cells (Fig. 3A). Similarly, infection of SUM-159 cells with a vector (5R) that expresses a c-erbB2-specific single-chain antibody, which has been shown to block ErbB2 function (19), diminished Ras activity in serum-deprived cells (Fig. 3B). Neither AG1478 treatment nor 5R expression had a significant effect on the apoptotic rate of SUM-159 cells (data not shown). To examine the effects of EGFR on invasion of β4-Scr cells, AG1478 treatment followed by invasion assay was done. As shown in Fig. 3C, AG1478 decreased cell invasion by ∼50%. Blocking of ErbB2 function by 5R expression also resulted in a significant decrease of β4-Scr cell invasion (Fig. 3D).

The α6β4 integrin “transactivates” EGFR phosphorylation. The finding that both EGFR/ErbB2 and α6β4 contribute to Ras activation in serum-deprived cells suggested a relationship between growth factor receptor and integrin signaling in this process. To test this possibility, we treated β4-Scr and β4 shRNA cells with EGF and did Ras assay. As shown in Fig. 4A, Ras activity was not stimulated further by EGF in serum-deprived control cells. Exogenous EGF, however, increased Ras activity >2-fold in the β4 shRNA cells. The finding that exogenous EGF did not increase Ras activation in cells that express α6β4 supports the possibility that α6β4 is “transactivating” EGFR signaling to some extent in serum-depleted condition. To test this hypothesis, we examined the tyrosine phosphorylation of the EGFR, which is indicative of receptor activation, as a function of α6β4 expression. Indeed, as shown in Fig. 4B, depletion of β4 expression resulted in an ∼50% decrease in EGFR phosphorylation in comparison with control cells. As predicted from our previous data, this effect of α6β4 on EGFR phosphorylation was only seen in serum-deprived cells (Fig. 4B). Of note, α6β4 had no effect on the expression of the EGFR in SUM-159 cells (Fig. 4B). The specificity of the effect of α6β4 expression on EGFR phosphorylation is evidenced by the finding that expression of β4 in the β4 shRNA cells increased EGFR phosphorylation ∼2-fold (Fig. 4B).

Phosphorylation of the EGFR is also dependent on ErbB2 signaling in SUM-159 cells. Inhibition of ErbB2 function with the 5R antibody resulted in a marked decrease in EGFR phosphorylation (Fig. 5A).

The α6β4 integrin can influence ErbB2 translation. We next examined the relationship between ErbB2 and EGFR in β4-Scr and β4 shRNA cells by coimmunoprecipitation. As shown in Fig. 5B, depletion of α6β4 expression resulted in a loss of direct and indirect interaction of ErbB2 and EGFR. More specifically and surprisingly, a significant reduction in ErbB2 expression was evident in the β4 shRNA cells relative to the control cells (Fig. 5B). This finding, which was confirmed in a separate immunoblotting experiment (Fig. 5C), prompted us to examine the relationship between α6β4 and ErbB2 expression more rigorously. Reexpression of β4 in the β4 RNAi cells increased ErbB2 expression (Fig. 5C), a finding that supports the specificity of the shRNA results. We further studied the relationship between α6β4 integrin and ErbB2 expression in another breast carcinoma cell line, MDA-MB-231. As shown in Fig. 5D, β4 shRNA cells exhibited a significant reduction in β4 integrin as well as ErbB2.

Although a decrease in ErbB2 protein expression was evident in the β4 RNAi cells, no significant changes in erbB2 mRNA were apparent as assessed by Northern blot analysis (Fig. 6A). These findings suggested that α6β4 may influence the translation of erbB2 in SUM-159 cells. Given that the concentration of eIF4E is rate-limiting for Cap-dependent translation, we reduced eIF4E expression in SUM-159 β4 control and β4 shRNA cells using an antisense oligonucleotide. This approach reduced eIF4E expression by ∼60% compared with cells that expressed the sense oligonucleotide, and it resulted in a concomitant decrease in phospho-ErbB2, ErbB2, and phospho-EGFR expressions in β4 control cells (Fig. 6B). eIF4E reduction in β4 shRNA cells, however, did not affect those protein levels (Fig. 6B), suggesting that β4 shRNA cells may already have low eIF4E activity. To test this hypothesis, we examined phospho-eIF4E level in the β4 shRNA and control cells. As shown in Fig. 6C, β4 shRNA cells exhibited lower phospho-eIF4E level that was consistent with lower ErbB2 level in these cells. To obtain more definitive evidence that α6β4 is regulating erbB2 translation, we did polysome analysis of the erbB2 message. mRNA isolated from the β4 shRNA and control cells was fractionated on a sucrose gradient and the relative expression of erbB2 mRNA in each fraction was determined by Northern blotting. As shown in Fig. 6D, a striking difference in the distribution of erbB2 mRNA was evident in the two populations of cells. In the control cells, erbB2 mRNA fractionated in the heavy polysomal region. In contrast, in the β4 shRNA cells, the majority of erbB2 mRNA showed a shift toward the light polysomal region. This result strongly supports the hypothesis that α6β4 can regulate the cap-dependent translation of erbB2 in SUM-159 cells. We next examined the involvement of eIF4E in cell invasion. As shown in Fig. 6E, the reduction of eIF4E expression resulted in an ∼45% decrease in invasion.

The involvement of integrin α6β4 in tumor cell survival, migration, and invasion is well established. Clinical studies also support the positive correlation between integrin α6β4 and tumor progression and poor clinical outcome (20, 21). An emerging consensus is that α6β4 synergizes with specific molecules such as ErbB2, EGFR, Ron, Fyn, c-Met, protein kinase C (PKC), CD151, and CD9 to activate key signaling pathways for the invasion and migration of carcinoma cells (14, 22). The most studied signaling molecule activated by integrin α6β4 is PI3K, an important molecule for carcinoma invasion. For instance, integrin α6β4 functions together with ErbB2, c-Met, and insulin receptor substrates to activate PI3K (14, 23). This PI3K, in turn, affects clustering of integrin α6β4 thereby increasing tumor invasion (24), suggesting a positive feedback loop between α6β4 and PI3K. In addition to the stimulation of PI3K signaling, the α6β4 integrin has also been shown to promote migration by the activation of other signaling molecules such as Ras and RhoA GTPase, Rac, mitogen-activated protein kinase pathways (14, 23). However, not much is known about the roles of this integrin on the expression of genes that are important for the carcinoma invasion and migration. It has been shown that α6β4 integrin regulates the activities of nuclear factor of activated T cells and nuclear factor κB transcription factors (25, 26). The ability of the α6β4 integrin to signal to transcriptional factors suggests that this integrin could regulate the transcription of genes. Recently, it has been shown that integrin α6β4 promotes transcription of autotaxin, an important autocrine motility factor in breast carcinoma cells, via the nuclear factor of activated T cell–dependent pathway (27). Little is known about the roles of this integrin on translation. Only one report shows that integrin α6β4 stimulates vascular endothelial growth factor (VEGF) translation via mammalian target of rapamycin (mTOR)–dependent inactivation of 4E-binding protein 1 (28). Here, we reveal that integrin α6β4 regulates the activity of Ras, a key player in cell invasion, and this is achieved by regulating ErbB2 translation in serum-depleted cells. All these data indicate that integrin α6β4 can interact and/or modulate specific molecules to synergistically activate signal molecules, which in turn regulate transcription and translation of molecules that are important for carcinoma invasion and migration.

Ras proteins control key signaling pathways that are involved in cell invasion and metastasis (8). Ras mutation can be found in 90% of pancreatic cancers, 50% of colon cancers, 50% of thyroid cancers, and 5% of breast cancers (7). Low percentages of ras mutations are found in breast cancers; however, Ras proteins are active in breast tumors by highly activated upstream signaling molecules, growth factor receptors (29). EGFR and ErbB2 overactivation is the most common reason for Ras pathway activation in many cancer cells (8). ErbB2 is a very potent oncoprotein (30). It is overexpressed in ∼30% of invasive breast cancer cases and other cancers and is associated with enhanced tumorigenecity, metastatic potential, a poor prognosis, and resistance to chemotherapy (30). Recent reports show that ErbB2 up-regulates the chemokine receptor CXCR4 and also inhibits degradation of CXCR4 thereby increasing tumor metastasis (31). ErbB2 has been an important target in breast cancer therapy where treatment with the anti-ErbB2 monoclonal antibody trastuzumab (Herceptin) has proved effective (32). Therefore, an understanding of the mechanisms that regulate ErbB2 has clinical importance. Regulation of ErbB2 expression by integrin α6β4 suggests a novel mechanism by which this integrin functions in the regulation of growth factor receptor activity. Previous studies on the relationship between growth factors and integrin α6β4 have shown that growth factors regulate integrin α6β4 functions (1). For example, epidermal growth factor (EGF) stimulates phosphorylation of the β4 subunit on serine residues via PKCα thereby inducing hemidesmosomes disassembly (33). EGF also activates one of the Src family kinase, Fyn, which in turn phosphorylates tyrosine residues of integrin β4 cytoplasmic domain and induces cell migration (1). In addition, integrin α6β4 is necessary for EGF-induced Rac activity and Rac mediates tumor survival (14). Hepatocyte growth factor also has been shown to induce tyrosine phosphorylation of the integrin β4, which in turn recruits Src homology and collagen (protein) and PI3K, thereby increasing cell migration (1). These studies suggest that integrin α6β4 plays its role as a mediator when it is activated by growth factors. In contrast, our data provide evidence that α6β4 integrin regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. Of interest, integrin α6β4 had no effect on Ras activity, ErbB2 expression, and cell invasion in the presence of serum but has profound effects on these in the absence of exogenous growth factor stimulation. Given the facts that Ras and ErbB2 play key roles in tumor cell invasion and metastasis, and carcinomas express elevated levels of integrin α6β4 (1, 4), our studies suggest the possibility of integrin α6β4 for facilitating tumor invasion in growth factor–deprived circumstances.

The involvement of integrin α6β4 on the control of ErbB2 expression through eIF4E suggests essential roles of translation regulation by this integrin in tumor invasion. Although transcriptional regulation is crucial, the flow of information from genes to proteins is too slow to accommodate rapid changes in the environment (34). Cells maintain a pool of mRNA and modulate the rates of key protein levels by a variety of sophisticated means such as translational controls. Especially, tumor cells have to escape the region that is devoid of nutrients and they encounter a variety of environments during metastasis. Therefore, tumor cells develop an efficient and powerful translational machinery by several mechanisms: aberrantly activated signal transduction pathways, changes of the expression or availability of translational machinery components, and/or variations in mRNA sequences (35). The key component for translation is eIF4E. This functions in the rate-limiting steps of translation initiation, and phosphorylation of eIF4E increases the rate of protein synthesis during tumor growth, invasion, and metastasis (34, 35). Most mRNAs are not affected by low eIF4E but some proteins, such as VEGF, c-myc, ornithine decarboxylase, and cyclin D1, are poorly translated when free eIF4E is limiting (35). The major regulators of eIF4E are Ras and Akt/mTOR (36, 37). Integrin α6β4 regulates eIF4E, Ras, and Akt/mTOR; therefore, this integrin may function as a major regulator for translation in carcinomas. Of interest, the SUM-159 cells that we used expressed low levels of integrin β4; however, these cells exhibited a great effect on ErbB2 translation and Ras activity. This suggests that even low levels of this integrin can function as a major translation regulator.

Because Ras and Akt/mTOR are activated by molecules such as integrin α6β4 in many carcinomas, a good correlation is often observed among relative protein level, mRNA level, and gene copy number in tumors. However, many carcinomas have proteins that do not correlate with gene copy number and mRNA abundance. In the case of ErbB2, erbB2 gene amplification or mRNA overexpression has been thought to cause the elevated expression of the ErbB2 protein (38). However, the level of erbB2 mRNA is not always proportional to the gene copy number, nor does the abundance of ErbB2 protein always reflect the mRNA levels. Therefore, some reports suggest the possibility of posttranscriptional regulation of erbB2 (39, 40) but no reports have identified this. Our finding that integrin α6β4 regulates ErbB2 protein level through eIF4E suggests a novel mechanism for ErbB2 overexpression in tumor cells and new roles of this integrin for translational regulation. Taken together, it can be said that ErbB2 overexpression is regulated by complex and multiple levels: gene copy number, transcriptional, and translational control. Therefore, tumor cells that have an elevated level of erbB2 mRNA coupled with efficient translational machinery, which is strengthened by molecules such as integrin α6β4, would benefit the most for ErbB2-dependent carcinoma invasion.

Here, we provided mechanistic understanding of integrin α6β4-mediated breast carcinoma invasion. A potentially important consequence of our work and other reports is that integrin α6β4 may be an attractive therapeutic target for breast cancer, especially in invading and metastasizing cells. It could be an ideal target in terms that it is a cell-surface receptor and exists with other growth factor receptors and cell-surface molecules as complexes that influence carcinoma invasion. In this regard, development of function-blocking anti-β4 antibodies could offer a novel treatment for breast cancer patients.

Grant support: NIH grant CA 80789.

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 Drs. Arthur M. Mercurio and John Blenis for their helpful insight and discussion and Drs. Nancy E. Hynes, Arigo De Benedetti, Johannes L. Bos, and Kun-Liang Guan for providing pBABE-5R, eIF4E-sense and eIF4E-antisense constructs, GST-Raf1-RBD, and DN-H-Ras plasmids, respectively.

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