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J. Biol. Chem., Vol. 283, Issue 8, 5127-5137, February 22, 2008

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Cdc42 Regulates E-cadherin Ubiquitination and Degradation through an Epidermal Growth Factor Receptor to Src-mediated Pathway^*

From the Division of Monoclonal Antibodies, Office of Biotechnology Products, Office of Pharmaceutical Science, Center for Drug Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland 20892

Received for publication, April 19, 2007 , and in revised form, October 31, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
E-cadherins play an essential role in maintaining epithelial polarity by forming Ca²⁺-dependent adherens junctions between epithelial cells. Here, we report that Ca²⁺ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca²⁺ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca²⁺ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Adherens junctions are one of the major structures responsible for cell-cell adhesion between epithelial cells, and their formation is required for the establishment of a differentiated epithelium (1). Adherens junctions form by Ca²⁺-dependent homophilic interactions between neighboring cells mediated by type I receptors known as cadherins (2, 3). Adherens junctions are dynamic structures that are remodeled as needed based on cellular conditions such as during embryonic development and wound healing. Remodeling of adherens junctions requires endocytosis and recycling of E-cadherin (4). During epithelial tumor progression, disruption of adherens junctions and decreased E-cadherin expression occurs and contributes to epithelial to mesenchymal transitions (EMT)⁴ (5). Although sustained repression of E-cadherin occurs at the transcriptional level, initial decreases in E-cadherin levels likely occur by lysosomal degradation (6, 7). Mechanisms regulating the trafficking of E-cadherin for lysosomal degradation are poorly understood.

Overexpression of the epidermal growth factor receptor (EGFR) correlates with metastasis in breast cancer and other carcinomas. EGF stimulation of epithelial cells has previously been shown to disrupt adherens junctions (8), and chronic stimulation causes up-regulation of E-cadherin transcriptional repressors such as TWIST and Snail (9, 10). Conversely, inhibition of EGFR signaling contributes to increased E-cadherin protein levels and restoration of adherens junctions in lung cancer and breast cancer cell lines (11, 12). Although it is clear that increased EGFR signaling contributes to sustained repression of E-cadherin expression, it is not known whether or not EGFR contributes to the initial down-regulation of E-cadherin by lysosomal degradation.

Src, a non-receptor tyrosine kinase, is a central regulator of signaling downstream of EGFR and has been shown to regulate EMT by disrupting adherens junctions (13, 14). Mechanistically, Src alters E-cadherin trafficking by redirecting E-cadherin from a recycling pathway to a lysosomal-targeting pathway (7). In Madin-Darby canine kidney cells Src phosphorylation of E-cadherin leads to binding of the E3 ligase Hakai; Hakai then ubiquitinates E-cadherin, resulting in E-cadherin endocytosis (15). The downstream effect of Hakai-mediated E-cadherin ubiquitination and endocytosis has not been characterized.

Rho family GTPases, including Cdc42, Rac1, and RhoA, have all been found to regulate adherens junctions (16, 17). The majority of data indicate that Rho GTPases contribute to the establishment and stabilization of adherens junctions in mouse L fibroblasts expressing E-cadherin (EL cells) (18, 19). For instance, Cdc42 and Rac1 stabilize adherens junctions in Madin-Darby canine kidney cells, and activation of both contributes to the initial formation of adherens junctions (20-22). It has been recently reported that FRG, a Cdc42-specific guanine nucleotide exchange factor, regulates cell junction assembly through activation of Cdc42 in Madin-Darby canine kidney cells (23). However, there are also data that suggest Rac1 and Cdc42 contribute to dissolution of adherens junctions in human epidermal keratinocytes (24, 25). For Rac1, disruption of adherens junctions is in part mediated by stimulating endocytosis of E-cadherin (25). Overexpression of constitutively active Cdc42 was also found to prevent adherens junction formation in keratinocytes, suggesting that Cdc42 may negatively regulate adherens junction establishment (26).

Activation of Cdc42 in fibroblast cells has been reported to inhibit Cbl-mediated ubiquitination and degradation of EGFR, resulting in cellular transformation due to the accumulation of EGFR and sustained EGF-stimulated ERK activation (27). In MDA-MB-231 breast cancer cells, down-regulation of Cdc42 restores EGFR degradation and decreases cell migration (28). Based on this information, we hypothesized that Cdc42 might regulate E-cadherin degradation through an ubiquitin-mediated pathway to dissolute adherens junctions and positively regulate cell migration. Given the roles of Cdc42, Src, and EGFR in adherens junction dynamics, we addressed the question of whether signaling through Cdc42, EGFR, and Src cooperatively contributes to the dissolution of adherens junctions, leading to E-cadherin ubiquitination and degradation.

Here, we provide evidence that after endocytosis induced by Ca²⁺ depletion, E-cadherin is ubiquitinated and targeted for lysosomal degradation and that Cdc42 plays a critical role in regulating this process through an EGFR to Src signaling pathway. We demonstrate that Cdc42 is activated under Ca²⁺-free conditions. Activation of Cdc42 subsequently initiates the activation of EGFR and Src. Activated Src in turn tyrosine-phosphorylates E-cadherin, leading to Hakai-mediated E-cadherin ubiquitination. These data are supported by the results that silencing Cdc42 inhibits activation of EGFR and Src, resulting in a reduction in E-cadherin degradation. When Cdc42 is constitutively activated, as is the case for Cdc42(F28L), an oncogenic mutant that undergoes spontaneous activation (29, 30), the persistent activation of EGFR and Src leads to a significant enhancement in E-cadherin ubiquitination and degradation, causing the dissolution of E-cadherin-based adherens junctions and an increased rate of cell migration. Furthermore, our data indicate that activated Cdc42 binds to E-cadherin in a GTP-dependent manner, and the binding of Cdc42 is essential for Cdc42 to induce the dissolution of adherens junctions.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Antibodies—Antibodies against E-cadherin (clone 36), EGFR, and activated EGFR were from BD Transduction Laboratories. Antibodies against the N terminus of E-cadherin (clone HECD) and against v-Src (mouse monoclonal antibody 327) were from Calbiochem. Anti-HA antibody was from Covance (Richmond, CA). Rabbit anti-Src (36D10) was from Cell Signaling (Beverly, MA). Anti-Hakai and anti-ubiquitin antibodies were from Zymed Laboratories Inc. (South San Francisco, CA). Anti-phosphotyrosine (clone 4G10) antibody was from Upstate%20Biotechnology">Upstate Biotechnology, Inc., (Lake Placid, NY). Anti-LAMP1 (H4A3) antibody, developed by J. Thomas August and James E. K. Hildreth, was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Alexa Fluor-conjugated secondary antibodies were from Invitrogen Molecular Probes (Eugene, OR).

Cell Culture and Transient and Stable Transfections—MCF-7 cells, from American Type Culture Collection (Manassas, VA), were grown in DMEM supplemented with 10% fetal bovine sera (FBS) and antibiotic/antimycotic. Cell culture media, trypsin, Lipofectin transfection reagent, and PLUS reagent were from Invitrogen). FBS was from Omega Scientific, Inc. (Tarzana, CA). Ca²⁺-free DMEM was from Invitrogen. Cells stably expressing Cdc42(F28L) were generated using methods described previously (29, 30). Transient transfections using Lipofectin and PLUS reagent were done according to the manufacturer's protocol. SMARTpool short interfering RNA (siRNA) targeting human Cdc42, human Hakai, and control non-targeting siRNA were purchased from Dharmacon (Chicago, IL). MG132 proteasome inhibitor was purchased from Calbiochem. Tyrphostin AG1478 was purchased from Sigma-Aldrich. For EGFR inhibition studies, cells were seeded in the morning and allowed to attach and spread for 8 h. Me₂SO (control) or AG1478 (at 10 µM) was then added overnight. Cells were then switched to Ca²⁺-free DMEM in the presence of Me₂SO or AG1478 (10 µM) for the indicated times.

E-cadherin Ubiquitination and Degradation—MCF-7 cells were seeded overnight in 10% FBS/DMEM, and then cells were rinsed twice with phosphate-buffered saline before switching to Ca²⁺-free DMEM. At the indicated times cells were harvested, and whole cell lysates (WCL) were subjected to immunoprecipitation using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by Western blot with anti-ubiquitin antibody. The immunoprecipitated E-cadherin as indicated above or protein levels of E-cadherin in WCL harvested at the indicated time points were detected by Western blot using anti-E-cadherin antibody for assessing E-cadherin degradation.

E-cadherin Internalization and Recycling Assays—Internalization and recycling assays were done as previously described (4, 31). MCF-7 cells and Cdc42(F28L)-expressing MCF-7 (clone 19) cells were seeded overnight at 1.5 x 10⁶ cells/35-mm dish in complete media. Cells were biotinylated using EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer's protocol (Pierce). For recycling assays, accumulation of biotinylated proteins in early or sorting endocytic compartments was accomplished by incubating cells for 2 h at 18 °C (32-34). Cell surface biotinylated proteins were then stripped as previously described (35), and cells were incubated at 37 °C for the indicated times. Protein concentrations were determined using Bradford reagent (Bio-Rad). Biotinylated proteins from radioimmune precipitation assay buffer-extracted WCL, and media were affinity-precipitated using immobilized NeutrAvidin^TM protein per the manufacturer's protocol (Pierce). Levels of biotinylated E-cadherin were determined by Western blot using anti-E-cadherin (HECD) antibody.

p21 Binding Domain (PBD) Assay—The determination of the levels of GTP-bound Cdc42 were done using the GST-PBD of p21-activated kinase (PAK) as described previously (36).

Immunofluorescence—Cells were seeded overnight on chamber slides at 1 x 10⁵ cells/well in 10% FBS/DMEM. Cells were then rinsed two times with phosphate-buffered saline and incubated for the indicated times in DMEM or Ca²⁺-free DMEM. Cells were fixed and stained as previously described (28). Actin was detected using rhodamine-conjugated phalloidin (Invitrogen Molecular Probes). Src was detected using rabbit anti-Src (36D10).

Migration Assays—Boyden chamber migrations assays, fixation, and staining were done as described previously (37). Cells were incubated for 5 h at 37 °C with 5% CO₂ and then fixed and stained. Wound healing assays are described in the Fig. 2E legend.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. E-cadherin is ubiquitinated and degraded after the switch to Ca2+-free media. A, MCF-7 cells were seeded overnight in 10% FBS/DMEM and then switched to DMEM without Ca2+ for the indicated times. WCL were subjected to immunoprecipitation using anti-E-cadherin antibody. E-cadherin was detected by Western blot with an anti-E-cadherin antibody (upper blot). Arrows, E-cadherin degradation products. Actin Western blot analysis was done on WCL to confirm that equal amounts of protein were used for immunoprecipitations (lower blot). B, MCF-7 cells were grown as described in A and then switched to DMEM without Ca2+ for 3 h. WCL were subjected to immunoprecipitation (IP) using anti-E-cadherin antibody or control IgG. Ubiquitinated (Ubi) E-cadherin was detected by Western blot with an anti-ubiquitin antibody (upper blot). E-cadherin in WCL was detected by Western blot using antibody against E-cadherin (middle blot). Arrows, E-cadherin degradation products. Actin Western blot analysis was done on WCL to confirm that equal amounts of protein were used for immunoprecipitations (lower blot). C, MCF-7 cells were transfected with a plasmid encoding HA-tagged ubiquitin. Cells were then incubated in DMEM with or without Ca2+ for 3 h 48 h post-transfection. WCL were subjected to anti-E-cadherin antibody or IgG control antibody immunoprecipitations. Ubiquitinated E-cadherin was detected by blotting with anti-HA antibody. E-cadherin in WCL was detected by Western blot using antibody against E-cadherin (middle blot). Arrows, E-cadherin degradation products. Actin Western blot analysis was done on WCL to confirm that equal amounts of protein were used for immunoprecipitations (lower blot). D, MCF-7 cells were seeded overnight in 10% FBS/DMEM and then switched to DMEM without Ca2+ in the presence or absence of the proteasomal inhibitor MG132 at 25 µgml-1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by Western blot with an anti-ubiquitin antibody (top panel). The blot was then stripped and reprobed for E-cadherin (second from top panel). E-cadherin in WCL was detected by Western blot using antibody against E-cadherin (third from top panel). Arrows, E-cadherin degradation products. Actin Western blot analysis was done to confirm equal protein loading (bottom panel). E, MCF-7 cells were seeded as described in A and then switched to DMEM with or without Ca2+ for 1 h. Cells were stained for E-cadherin (green) and LAMP-1 (red). Arrows, lysosomes. Magnification, 1200x. Graph, percentage of cells with colocalization between E-cadherin and LAMP-1. A minimum of 100 cells was counted per condition per experiment. Data are the mean ± S.D. of three independent experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We set out to investigate whether endocytosed E-cadherin induced by Ca²⁺ depletion can be targeted for lysosomal degradation. Cells were switched to Ca²⁺-free media to initiate E-cadherin endocytosis and harvested at the indicated time points, and then E-cadherin proteins were immunoprecipitated from WCL. Western blotting was used to detect E-cadherin protein levels (Fig. 1A). A significant reduction in E-cadherin protein levels was observed after a 3-h incubation in Ca²⁺-free media. Two lower molecular weight protein bands that are likely E-cadherin degradation products were also recognized by the anti-E-cadherin antibody after incubation of cells in Ca²⁺-free media (Fig. 1A, arrows).

We next tested whether E-cadherin was ubiquitinated upon Ca²⁺ depletion. Significant ubiquitination of immunoprecipitated E-cadherin was observed after a 3-h Ca²⁺ depletion (Fig. 1B, compare lanes 1 and 2, upper image), and E-cadherin ubiquitination was not detected when control IgG was used for immunoprecipitation (lanes 3 and 4). Decreased total protein levels of E-cadherin and the two lower protein bands were also detected in WCL upon Ca²⁺ depletion (lanes 2 and 4 in Fig. 1B, middle image). To confirm the ubiquitination of E-cadherin under Ca²⁺-free conditions, we transiently expressed HA-ubiquitin in MCF-7 cells. The experimental procedures for Fig. 1C were essentially the same as described in Fig. 1B, except the immunoprecipitated E-cadherin was detected by anti-HA antibody. As shown in Fig. 1C, Ca²⁺ depletion significantly induced E-cadherin ubiquitination that was not detected when control IgG was used for immunoprecipitation. To elucidate the role of the proteasome in Ca²⁺ depletion-induced E-cadherin ubiquitination and degradation, we treated cells with MG132, a proteasomal inhibitor. Consistent with the results shown in Fig. 1, B and C, upon Ca²⁺ depletion, E-cadherin was ubiquitinated and degraded (Fig. 1D, lane 3, top three images). However, treatment of cells with MG132 did not prevent E-cadherin degradation but appeared to increase its ubiquitination and degradation (lane 4 in Fig. 1D, top three images), suggesting that Ca²⁺ depletion induced E-cadherin degradation may occur in lysosomes.

We next determined if E-cadherin was transported to lysosomes. As shown in Fig. 1E, E-cadherin colocalized with the lysosomal marker LAMP-1 in a perinuclear compartment after a 1-h Ca²⁺ depletion, whereas there was no significant colocalization between E-cadherin and LAMP-1 when cells were cultured in Ca²⁺-containing media. After 1 h of incubation in Ca²⁺-free media, 20% of cells had E-cadherin colocalization with LAMP-1 as compared with 5% of cells grown in Ca²⁺ containing media (Fig. 1E, graph). Taken together, we conclude that E-cadherin ubiquitination can be induced by Ca²⁺ depletion, and ubiquitinated E-cadherin is targeted for lysosomal degradation.

Ca²⁺ Depletion Triggers the Activation of Cdc42 and Enhances the Rate of Cell Migration—Under Ca²⁺-free conditions, we observed that MCF-7 cells gradually dissociated, depolarized, and formed numerous cellular protrusions (Fig. 2A, right panels). Results from staining cells with rhodamine-conjugated phalloidin to detect actin suggested that the cellular protrusions observed under Ca²⁺-free conditions were microspikes or filopodia (Fig. 2B). It is well established that activation of Cdc42 triggers the formation of filopodia (38). We then asked whether Ca²⁺ depletion induced the activation of Cdc42. A GST-PBD pulldown assay provided evidence that levels of activated Cdc42 were elevated under Ca²⁺-free conditions (Fig. 2C, compare 0 h to 1 and 3 h). Taken together, these data suggest that the Ca²⁺-free media-induced filopodium formation might be the direct consequence of Cdc42 activation. Activation of Cdc42 has been implicated in the regulation of cell migration (38). We next tested whether depletion of Ca²⁺ could increase the rate of cell migration. As measured by Boyden chamber and wound healing migration assays, cell migration in Ca²⁺-free media was significantly increased as compared with that in Ca²⁺ containing media (Fig. 2, D and E).

View larger version (76K): [in this window] [in a new window]   FIGURE 2. Disengagement of cell-cell adhesions by Ca2+ depletion activates Cdc42 and enhances cell migration. A, MCF-7 breast cancer cells were seeded overnight in 10% FBS/DMEM and then switched to DMEM with or without Ca2+ for the indicated times. Arrows, cellular protrusions. Magnification, 200x. B, MCF-7 cells were incubated for 3 h in DMEM with or without Ca2+. Cells were then fixed, permeabilized, and stained with rhodamine-conjugated phalloidin to detect F-actin. Magnification, 2400x. C, MCF-7 cells were incubated in Ca2+-free media for the indicated times. Activated Cdc42 was affinity-precipitated from WCL by incubating with GST-PBD-agarose. Bound Cdc42 was detected by Western blot for Cdc42. D, migration of MCF-7 cells in 0.1% BSA in Ca2+-containing or Ca2+-free DMEM was determined using a Boyden chamber migration assay. 12 µg/ml fibronectin was used as the chemoattractant in the lower chamber. Upper and lower chambers were separated by an 8.0 µm polycarbonate membrane. 5 h after seeding cells were fixed in methanol and then Giemsa-stained. Data are the mean ± S.D. of three independent experiments. E, wound healing assays were done on MCF-7 cells seeded overnight in DMEM medium supplemented with 10% FBS in 12 well plates. Cells were then scratch-wounded across the cell monolayer using a 1000-µl micropipette and grown in DMEM with or without Ca2+. Phase contrast images were taken at the indicated times. Magnification, 200x.

We next tested whether activation of Cdc42 led to the dissolution of E-cadherin-based adherens junctions. Although transient expression of wild-type Cdc42 and dominant negative Cdc42(T17N) had no effect on E-cadherin localization at adherens junctions (Fig. 3D, top and bottom panels), expression of Cdc42(Q61L) produced filopodium formation and induced the disappearance of E-cadherin-based adherens junctions (Fig. 3D, middle panels). Adherens junctions were then examined in the MCF-7 stables cell lines. MCF-7 vector control cells had robust colocalization between E-cadherin and actin at adherens junctions (Fig. 3E, left panels). In contrast, filopodia induced by the stable expression of Cdc42(F28L) interrupted E-cadherin-based adherens junctions (Fig. 3E, right panels). Taken together, these data indicate that expression of activated Cdc42 in MCF-7 cells disrupts the polarized epithelial morphology and contributes to the development of a mesenchyme-like morphology.

A strong correlation between loss of E-cadherin at the level of the cell surface and enhanced cell invasiveness has been described (39). We tested whether Cdc42(F28L) had the ability to increase cell migration using a Boyden chamber assay. MCF-7 cells stably expressing Cdc42(F28L) (clone 19) exhibited approximately a 2-fold increase in the rate of cell migration as compared with control MCF-7 cells (Fig. 3F), consistent with data shown in Fig. 2, D and E.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. Activation of Cdc42 increases E-cadherin ubiquitination and degradation and interrupts E-cadherin-based cell-cell adhesions. A, MCF-7 vector control cells (Vector) and MCF-7 cells stably expressing Cdc42(F28L) (clones 7, 13, and 19) were incubated for the indicated times in DMEM with or without Ca2+. The levels of E-cadherin in WCL were detected by Western blot using an anti-E-cadherin antibody. Actin Western blot analysis was done to confirm equal protein loading (bottom panel). B, WCL from vector control and each of the Cdc42(F28L)-expressing clones were Western-blotted with anti-HA antibody to detect HA-Cdc42(F28L) protein levels. C, MCF-7 vector control and Cdc42(F28L)-expressing MCF-7 cells were incubated in Ca2+-free DMEM for 3 h. E-cadherin was immunoprecipitated (IP) from WCL and probed for ubiquitin by Western blot (top panel). Actin Western blot analysis was done to confirm equal protein loading (bottom panel). D, MCF-7 cells were transiently transfected with the indicated HA-tagged Cdc42 mammalian expression vectors. Fixed and permeabilized cells were stained for HA (red) and E-cadherin (green). Arrows, cell-cell adhesions; open arrows, disrupted cell-cell adhesion. Magnification, 1200x. E, MCF-7 vector control (Vector) and MCF-7 stably expressing Cdc42(F28L) (clone 19) were stained for E-cadherin (green) and F-actin (red). Arrows, cell-cell adhesions; open arrows, disrupted cell-cell adhesions. Magnification, 1800x. F, migration of MCF-7 vector control and Cdc42(F28L)-expressing cells (clone 19) in 0.1% BSA in Ca2+-free DMEM was determined using a Boyden chamber migration assay as described in Fig. 2D.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Stable expression of Cdc42(F28L) increases the degradation of endocytosed E-cadherin and reduces E-cadherin recycling. A, MCF-7 vector control (Vector) and MCF-7 cells stably expressing Cdc42(F28L) (clone 19) cells were biotinylated on ice for 1 h; free biotin was then quenched. Cells were then incubated at 37 °C in Ca2+-free DMEM for the indicated times and then placed on ice. Cell surface biotinylation was removed by incubating cells on ice in glutathione stripping buffer. After stripping and protein extraction, total internalized biotinylated proteins were affinity-precipitated from WCL by incubating with avidin-conjugated agarose. Bound proteins were then resolved by SDS-PAGE. Biotinylated E-cadherin was detected by Western blot for E-cadherin (top panel). An actin Western blot was done to confirm equal protein loading (bottom panel). Data are representative of two independent experiments. B, MCF-7 vector control and MCF-7 cells stably expressing Cdc42(F28L) (clone 19) cells were biotinylated as described in A. Cells were then incubated for 2 h at 18 °C, which permits endocytosis but prevents trafficking beyond an early endosomal compartment. Cell surface biotinylation was then glutathione-stripped, and cells were incubated at 37 °C in Ca2+ containing DMEM for the indicated times. Cells were subjected to a mild trypsin digest (0.01%) to release the ecto domain of cell surface E-cadherin into the cell media. The cell media was then incubated with avidin-conjugated agarose to affinity precipitate biotinylated ecto domain of E-cadherin released from the cell surface by the trypsin digest (Ecto, lanes 4 and 5). Internalized biotinylated proteins of WCL were also affinity-precipitated using avidin-conjugated agarose. Bound proteins were resolved by SDS-PAGE followed by Western blot for E-cadherin using an anti-E-cadherin antibody that recognizes an N-terminal epitope. Lane 1, glutathione-stripped (GS) immediately after biotinylation. Lane 2, total biotinylation. Lane 3, t = 0 min total internalized after 2 h of incubation at 18 °C. Lanes 4 and 5, t = 30 and 60 min at 37 °C.

View larger version (36K): [in this window] [in a new window]   FIGURE 5. Ca2+ depletion induces activation of Src, and Cdc42(F28L) increases Src activity in MCF-7 cells. A, MCF-7 cells were grown in Ca2+-free DMEM media for the indicated times. WCL were subjected to either anti-phosphotyrosine antibody (clone 4G10) or nonspecific IgG control immunoprecipitations (IP) followed by Western blot analysis for activated Src using anti-phosphotyrosine 416 Src (SrcpY416) antibody. Blots were stripped and reprobed for Src using mouse anti-Src antibody (monoclonal antibody 327). B, MCF-7 cells were grown as described in A. WCL were then immunoprecipitated with either IgG control antibody or with anti-Src-Tyr(P)-416 antibody. Affinity precipitated Src-Tyr(P)-416 was detected with anti-Src-Tyr(P)-416. C, MCF-7 vector control cells and MCF-7 Cdc42(F28L) clone 19 cells were grown as described in A. WCL were subjected to anti-phosphotyrosine (clone 4G10) immunoprecipitations. Bound proteins were resolved by SDS-PAGE followed by Western blot analysis using antibodies against either Src (top panel) or Src-Tyr(P)-416 (third panel from top). The protein levels of actin in WCL were detected by Western blot analysis to confirm equal protein loading. D, levels of phosphorylated Tyr-416-Src were determined as described in C. A Western blot analysis of actin in WCL was determined to confirm equal protein loading.

Src Is Activated by Ca²⁺ Depletion in Cdc42(F28L)-overexpressing Cells—We next addressed whether Src was activated upon Ca²⁺ depletion. MCF-7 cells were incubated in DMEM with or without Ca²⁺ for 15 min, and WCL were subjected to immunoprecipitation using either an anti-phosphotyrosine antibody (clone 4G10) or control IgG. The immunoprecipitated proteins were analyzed by Western blot using an antibody against activated Src (Src-Tyr(P)-416). As shown in Fig. 5A, Src is activated upon Ca²⁺ depletion for 15 min (compare lane 1 and 2, top image). The control IgG heavy chains, which had a similar molecular weight to endogenous activated Src, were also recognized by anti-Src-Tyr(P)-416 antibody. To prove that control IgG did not non-specifically precipitate activated Src-Tyr(P)-416 from WCL, the Western blot was stripped and reprobed with an antibody against Src. Src was not detected in IgG control lanes 3 and 4 (Fig. 5A, bottom panel), indicating that IgG did not non-specifically precipitate activated Src from WCL. Consistent with data shown in the top panel of Fig. 5A, more Src was detected upon Ca²⁺ depletion (compare lane 1 to lane 2, bottom panel). We found the same to be true when anti-Src-Tyr(P)-416 antibody was used to immunoprecipitate activated Src from WCL (Fig. 5B). In vector control cells, Src was persistently activated under Ca²⁺-free conditions over a 15-min to 3-h time period (data not shown).

Because both Src and Cdc42 can be activated by Ca²⁺ depletion, we were compelled to determine whether or not stable expression of Cdc42(F28L) affected Src activation levels. When grown in empty DMEM containing Ca²⁺, Src activity was significantly higher in MCF-7 cells overexpressing Cdc42(F28L) (clone 19) than in vector control cells (Fig. 5C, compare lanes 1 and 3, first and third panels from the top). Additionally, no significant enhancement of Src phosphorylation was observed in clone 19 cells after Ca²⁺ depletion, indicating that Src was constitutively activated in Cdc42(F28L)-expressing cells (Fig. 5C, lanes 3 and 4). Similar results were obtained in the other Cdc42(F28L)-expressing cell lines (clone 13 and 7), where the activity of Src was also up-regulated (Fig. 5D). Taken together, these data provide evidence that the up-regulated Src activity was a result of activation of Cdc42 in cells.

We next determined if transient overexpression of wild-type Src (wt-c-Src) or constitutively active c-Src (Y527F-c-Src) affected adherens junction stability. Untransfected MCF-7 cells had robust adherens junctions as detected by E-cadherin staining (Fig. 6A, top panels). In contrast, cells with transient wt-c-Src or Y527-c-Src overexpression lacked adherens junctions (Fig. 6A, bottom two panels). Additionally, cells overexpressing Y527F-c-Src lacked epithelial cell polarity and had increased numbers of filopodia and pseudopodia similar to the morphologic changes induced by Ca²⁺ depletion or overexpression of activated Cdc42. Given that c-Src signals downstream of EGFR, we next tested whether activation of EGFR signaling leads to the dissolution of adherens junctions. Although transient expression of EGFP in MCF-7 cells had no effect on adherens junctions (Fig. 6B, top panels), overexpression of EGFP-EGFR in MCF-7 cells resulted in a dramatic morphological change, such that cells displayed elongated cellular protrusions, became depolarized, and lacked E-cadherin-based adherens junctions (Fig. 6B, bottom two panels).

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Effect of transient expression of Src or EGFR on E-cadherin-based adherens junctions in MCF-7 cells. A, MCF-7 cells were transfected overnight with either plasmids encoding either wild-type c-Src (wt-c-Src) or constitutively active c-Src (Y527F-c-Src) and then fixed and permeabilized. Cells were stained for Src using rabbit anti-Src antibody (clone 36D10) and E-cadherin using mouse anti-E-cadherin antibody. Alexa488-conjugated goat anti-rabbit and Alexa568-conjugated goat anti-mouse antibodies were used as secondary antibodies. UTF, untransfected cells. B, MCF-7 cells were transfected overnight with plasmids encoding either EGFP or EGFP-EGFR and then fixed and permeabilized. Cells were stained for E-cadherin (red). Arrows, adherens junctions; open arrows, disrupted adherens junctions. Magnification, 1200x.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Cdc42 is essential for the enhancement of EGFR signaling in MCF-7 cells. A, MCF-7 cells were cultured in DMEM for 12 h and were then incubated for 15 min in DMEM with or without Ca2+. WCL were subjected to immunoprecipitation using anti-phosphotyrosine antibody (4G10). Activated EGFR was detected by Western blot using an antibody against activated EGFR (top panel). MCF-7 vector control cells (Vector) and cells stably expressing Cdc42(F28L) (clone 19) were cultured overnight in DMEM and were then incubated for 15 min in DMEM with or without Ca2+. Activated ERK in WCL was detected using an antibody against phospho-44/42 ERK (p44/42 ERK) antibody (second and third panels from the top). The experimental procedure for the Cdc42 PBD-pulldown assay (bottom panel) was the same as described in Fig. 2C. B, MCF-7 vector control cells and MCF-7 Cdc42(F28L) cells (clones 19, 13, and 7) were cultured in DMEM supplemented with 10% FBS. Activated ERK1/2 (p44/42 ERK) and actin in WCL were detected by Western blot. C, MCF-7 cells were cultured in DMEM for 12 h and then incubated for 15 min in DMEM with or without EGF 100 ng ml-1 stimulation. Western blots were probed for activated ERK1/2 using anti-p44/42 ERK antibody. D, MCF-7 cells were grown overnight in DMEM and then incubated in Ca2+-free DMEM with or without EGF 100 ng ml-1 for the indicated times. WCL were Western-blotted for EGFR (top panel), E-cadherin (middle panel), and actin (bottom panel). E, MCF-7 cells stably expressing Cdc42(F28L) (clone 19) were cultured overnight in DMEM with (lane 3) or without (lanes 1 and 2) the EGFR kinase inhibitor, AG1478, at 10 µgml-1. Cells were then rinsed and incubated in DMEM media with or without Ca2+ for 0 and 3 h in the absence of (lanes 1 and 2) or 3 h in the presence of AG1478 at 10 µgml-1 (lane 3). WCL were Western-blotted for E-cadherin (top panel) and actin (bottom panel). F, MCF-7 cells were transfected for 48 h with control non-targeting siRNA (lanes 1 and 2), Cdc42-specific siRNA (lanes 3 and 4), or mock-transfected (lanes 5 and 6). Cells were then incubated for 15 min in DMEM with or without Ca2+. Tyrosine-phosphorylated proteins were immunoprecipitated (IP) from WCL with anti-phosphotyrosine antibody (clone 4G10) (lanes 1-4). Nonspecific protein precipitation was controlled by immunoprecipitation using an anti-HA antibody (lanes 5 and 6). After SDS-PAGE and transfer to polyvinylidene difluoride, Western blots were probed with anti-EGFR antibody (top panel) or anti-Src-phosphorylated on Y416 (Src-pY416) (second panel from top). Cdc42 (third panel from top) and actin (bottom panel) were detected by Western blot of WCL. G, MCF-7 cells were transfected for 48 h with control non-targeting siRNA (lanes 1 and 2) or Cdc42-specific siRNA (lanes 3 and 4). E-cadherin was immunoprecipitated from WCL and then Western-blotted with anti-E-cadherin antibody (top panel). WCL was Western-blotted for E-cadherin, Cdc42, and actin (bottom three panels).

We next assessed whether or not EGFR regulated E-cadherin degradation. We first determined if EGF treatment affected E-cadherin degradation. MCF-7 cells were incubated in Ca²⁺-free DMEM for the indicated times in the absence or presence of EGF (100 ng ml^-1). Increased E-cadherin degradation occurred when cells were incubated in the presence of EGF (Fig. 7D, compare lanes 2 and 3 to lanes 5 and 6). As expected, after EGF treatment, EGFR protein levels decreased. The same was true when MDA-MD-468 breast cancer cells, which express high levels of endogenous EGFR, were stimulated by EGF (data not shown). We next tested whether or not inhibiting EGFR activation using the EGFR inhibitor AG1478 decreased E-cadherin degradation after Ca²⁺ depletion. As shown in Fig. 7E, AG1478 was able to inhibit Ca²⁺ depletion-induced E-cadherin degradation in Cdc42(F28L)-expressing cells. Similar results were obtained in vector control cells (data not shown).

We then tested whether Cdc42 was necessary for the activation of EGFR and Src upon Ca²⁺ depletion. After transfection with either control non-targeting siRNA or Cdc42-targeting siRNA, tyrosine-phosphorylated EGFR and Src were immunoprecipitated using clone 4G10 anti-phosphotyrosine antibody. Activated EGFR and activated Src were then detected by Western blot (Fig. 7F). In cells transfected with control non-targeting siRNA, the levels of tyrosine-phosphorylated EGFR and Src were enhanced upon Ca²⁺ depletion (Fig. 7F, lanes 1 and 2, top two panels). In contrast, silencing Cdc42 blocked the activation of EGFR and Src induced by Ca²⁺ depletion (Fig. 7F, lanes 3 and 4, top two panels). Untransfected cells were subjected to an anti-HA immunoprecipitation to control for nonspecific protein precipitation.

We next determined if reducing Cdc42 protein levels affected E-cadherin degradation. MCF-7 cells were transfected with either control non-targeting siRNA or Cdc42-targeting siRNA. After Ca²⁺ depletion for 3 h, levels of E-cadherin were determined by E-cadherin Western blot of immunoprecipitated E-cadherin and of E-cadherin in WCL. After 3 h of incubation in Ca²⁺-free media, the level of E-cadherin in cells transfected with control siRNA was significantly decreased (Fig. 7G, compare lanes 1 and 2). However, silencing Cdc42 inhibited E-cadherin degradation induced by Ca²⁺ depletion such that the level of E-cadherin in cells transfected with Cdc42 siRNA was about 3-fold higher than that in cells transfected with control siRNA (Fig. 7G, compare lane 4 to lane 2), suggesting that Cdc42 activity is required for E-cadherin degradation induced by Ca²⁺ depletion.

Cdc42 Binds to E-cadherin and Hakai and Regulates Intracellular Localization of Hakai Proteins—The involvement of Cdc42 in the regulation of E-cadherin-based adherens junctions raised the possibility that Cdc42 might interact with E-cadherin. MCF-7 cells were transiently transfected with HA-tagged Cdc42 expression vectors, and then WCL were subjected to anti-HA immunoprecipitations. E-cadherin preferentially bound to constitutively activated Cdc42(Q61L) and Cdc42(F28L) (Fig. 8A, top panel). IQGAP1, which is a target protein of Cdc42 (41), was also found in the complex of activated Cdc42 and E-cadherin (data not shown). E-cadherin also associated with Cdc42(F28L) that was stably expressed in MCF-7 cells (Clone 19) (Fig. 8B). In GST pulldown assays, endogenous E-cadherin expressed in MCF-7 cells bound preferentially to constitutively activated Cdc42 (Cdc42(Q61L)) but not GST control or dominant negative Cdc42 (Cdc42(T17N)) (Fig. 8C). Taken together, these data indicate that Cdc42 interacts with E-cadherin in a GTP-dependent manner similar to the interaction between Cdc42 and its well established target protein, IQGAP.

Hakai has been reported to associate with E-cadherin and function as an E3 ligase that ubiquitinates E-cadherin (15). We first asked whether Hakai was responsible for the E-cadherin ubiquitination observed under Ca²⁺-free conditions. After a 48-h transfection with either control non-targeting siRNA (Fig. 8D, lanes 1, 2, 5, and 6) or Hakai-targeting siRNA (lanes 3, 4, 7, and 8), cells were switched to DMEM with or without Ca²⁺ for 3 h. Endogenous E-cadherin was immunoprecipitated using an anti-E-cadherin antibody, and the levels of ubiquitinated E-cadherin were assessed by Western blot. Silencing Hakai significantly reduced levels of E-cadherin ubiquitination induced by Ca²⁺ depletion in vector control cells as well as in Cdc42(F28L)-expressing cells (compare lane 2 to lane 4 and lane 6 to lane 8, respectively). The basal level of E-cadherin ubiquitination, as shown in Fig. 8D, in cells stably expressing Cdc42(F28L) was slightly elevated as compared with that in vector control cells (compare lanes 5 to 1). However, the levels of E-cadherin ubiquitination in Cdc42(F28L)-expressing cells was significantly higher than that in vector control cells upon Ca²⁺ depletion (compare lanes 6 to 2). Data in Fig. 8D also provide additional evidence that activated Cdc42 enhanced E-cadherin ubiquitination. The ability of Cdc42 to bind E-cadherin and to regulate E-cadherin ubiquitination and degradation led to the question of whether there was any functional linkage between Cdc42 and Hakai in cells. As shown in Fig. 8E, Hakai was found in a protein complex that contained activated Cdc42 and E-cadherin.

It has been reported that Hakai binds to E-cadherin that has been phosphorylated by Src and that after Hakai binding, E-cadherin is ubiquitinated (15). Given Src is activated upon Ca²⁺ depletion, we asked whether Ca²⁺ depletion affected E-cadherin tyrosine phosphorylation and whether or not Ca²⁺ depletion induced E-cadherin binding to Src or Hakai. After Ca²⁺ depletion, E-cadherin was tyrosine-phosphorylated (Fig. 8F, top panel). Additionally, E-cadherin, Hakai, and Src formed an immunocomplex in a Ca²⁺ depletion-dependent manner (Fig. 8F, bottom panels).

View larger version (49K): [in this window] [in a new window]   FIGURE 8. Cdc42 binds to E-cadherin and Hakai and regulates intracellular localization of Hakai in a GTP-dependent manner. A, MCF-7 cells were transiently transfected with the indicated HA-tagged Cdc42 constructs. WCL were immunoprecipitated (IP) with anti-HA antibody. Western blots were probed for E-cadherin (upper panel) and HA-tagged Cdc42 (middle panel). WCL were probed for E-cadherin to determined equal protein loading (bottom panel). B, WCL of MCF-7 vector control cells (Vector) and MCF-7 cells stably expressing Cdc42(F28L) (clone 19) were immunoprecipitated using anti-HA antibody. Western blots were probed for E-cadherin (top panel) and HA (bottom panel). C, MCF-7 cell WCL was incubated with either GST (lane 2) or recombinant GST-Cdc42 wt, GST-Cdc42(Q61L), or GST-Cdc42(T17N). Affinity-precipitated E-cadherin was detected by Western blot (top panel). Recombinant protein levels were detected by Coomassie Blue staining (lower panels). D, MCF-7 vector control cells (Vector) and Cdc42(F28L) expressing cells (clone 19) were transfected using control siRNA (lanes 1, 2, 5, and 6) or Hakai siRNA (lanes 3, 4, 7, and 8) for 48 h. Cells were then incubated for 3 h in DMEM with or without Ca2+. Cell lysates were subjected to immunoprecipitation using anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by Western blot with anti-ubiquitin antibody (upper panel). Endogenous Hakai was examined by Western blot using anti-Hakai antibody (middle panel). About 65% knockdown of Hakai protein was detected. Actin Western blot analysis was done to confirm equal protein loading (bottom panel). E, MCF-7 cell WCL was incubated with recombinant GST and GST-Cdc42(Q61L). Affinity-precipitated proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Blots were probed for E-cadherin and Hakai. Recombinant protein levels were detected by Coomassie Blue staining (lower panels). F, MCF-7 cells were incubated in DMEM with or without Ca2+ for 15 min. Immunoprecipitates from WCL were done using anti-phosphotyrosine antibody (4G10) (top panel) or anti-Src antibody (bottom three panels). Phosphorylated E-cadherin was detected by Western blot using an anti-E-cadherin antibody (top panel). Immunoprecipitated Src was detected by Western blot for Src (bottom panel). Co-immunoprecipitated E-cadherin and Hakai were detected using anti-E-cadherin and anti-Hakai antibodies. G, MCF-7 cells were transiently transfected with HA-tagged Cdc42(F28L), Cdc42(Q61L), or Cdc42(T17N), and cells were then fixed and stained for HA (red) and Hakai (green) 48 h post-transfection. Open arrows, Hakai localized in a perinuclear compartment. Magnification, 1800x. H, MCF-7 cells were incubated for 1 h in Ca2+-containing or Ca2+-free DMEM and then fixed and stained for E-cadherin (green) and Hakai (red). Arrows, colocalization of E-cadherin and Hakai (yellow in merged images). Magnification, 1800x. Right columns, enlargements of boxed regions.

Here, we describe a new role for Cdc42 in the regulation of E-cadherin-based adherens junctions. Based on a number of lines of evidence provided in this study, we propose a model for Cdc42-mediated regulation of E-cadherin ubiquitination and degradation (Fig. 9). In this model, Ca²⁺ depletion disengages homophilic interactions between E-cadherin and activates Cdc42. Activated Cdc42 then enhances EGFR signaling to mediate activation of Src. This leads to tyrosine phosphorylation of E-cadherin, which allows Hakai to bind to and subsequently ubiquitinate E-cadherin, thus resulting in E-cadherin lysosomal degradation. In cells overexpressing constitutively active Cdc42, the signaling through EGFR to Src is up-regulated, causing the enhancement in Hakai-mediated E-cadherin ubiquitination and targeting of E-cadherin for lysosomal degradation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cdc42 Regulates E-cadherin Ubiquitination and Degradation—During EMTs, E-cadherin-dependent adhesion is down-regulated, and cells lose their adhesive contact enabling cell migration (14, 42). Here, we show that loss of a polarized epithelial morphology and the acquisition of mesenchyme-like phenotype, which is accompanied by E-cadherin ubiquitination and lysosomal degradation, can be induced by Ca²⁺ depletion in MCF-7 cells. This indicates that a Ca²⁺ switching technique is a suitable method for studying signal transduction during the early stages of E-cadherin down-regulation and determining its biological relevance. Moreover, we demonstrate that overexpression of active Cdc42, Src, or EGFR causes morphological changes similar to that induced by Ca²⁺ depletion (Figs. 3 and 6) and that the rate of cell migration was increased in MCF-7 cells expressing Cdc42(F28L) as compared with vector control cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 9. Model depicting Cdc42-mediated regulation of E-cadherin ubiquitination and degradation. 1, Ca2+ depletion disengages homophilic interactions of E-cadherin proteins and activates Cdc42. 2 and 3, activated Cdc42 up-regulates EGFR signaling to mediate Src activation. 4, activated Src binds to and tyrosine-phosphorylates E-cadherin. 5 and 6, activated Cdc42 and Hakai associate with tyrosine-phosphorylated E-cadherin; Hakai ubiquitinates E-cadherin. 7, ubiquitinated E-cadherin undergoes endocytosis and is targeted for lysosomal degradation. When Cdc42 is constitutively activated, activities of EGFR and Src are up-regulated, resulting in enhanced Hakai-mediated E-cadherin ubiquitination followed by lysosomal degradation.

Several lines of evidence suggest functional connections between EGFR and E-cadherin (43). For instance, chronic EGF stimulation of epidermoid carcinoma cells initiates down-regulation of E-cadherin (10). Lowy and co-workers (40) reported that E-cadherin was found in a protein complex with EGFR, and Ca²⁺ depletion enhanced EGF-stimulated tyrosine phosphorylation of EGFR. We also found that the activity of EGFR is increased in Cdc42(F28L)-expressing cells, leading to the enhancement of E-cadherin ubiquitination and degradation. Furthermore, EGF stimulation increases E-cadherin degradation induced by Ca²⁺ depletion, whereas AG1478, a specific EGFR inhibitor, blocks E-cadherin degradation.

EGFR and ERK are activated when E-cadherin-based adherens junctions are disengaged by Ca²⁺ depletion. Silencing Cdc42 blocks EGFR activation, suggesting that Cdc42 may be responsible for this activation. However, the signaling pathway that mediates Ca²⁺ depletion-induced activation of Cdc42 still remains elusive. Recently, two Cdc42-specific guanine nucleotide exchange factors, FRG and Tuba, were found to regulate adherens junction assembly through activation of Cdc42 (23, 44). It will be interesting to investigate whether either of these Cdc42-specific guanine nucleotide exchange factors is responsible for the activation of Cdc42 upon Ca²⁺ depletion.

We identified E-cadherin as a specific binding partner of activated Cdc42. Although we do not know whether this interaction is direct or not, our data suggest that E-cadherin may function as a target molecule for Cdc42. We demonstrate that binding of Cdc42 to E-cadherin is essential for the actions of Cdc42 in adherens junctions. This is confirmed by the evidence that the dominant negative Cdc42T17N, which does not bind to E-cadherin, is unable to dissolute adherens junctions.

Given the importance of E-cadherin trafficking to the stability adherens junctions, a key question is how cells regulate the trafficking of internalized E-cadherin. The majority of previously published data indicate that internalized E-cadherin induced by Ca²⁺ depletion is either recycled to the plasma membrane or transiently sequestered inside the cell in sorting or recycling endosomes (4, 45). However, we found that after Ca²⁺ depletion endocytosed E-cadherin is ubiquitinated and targeted for lysosomal degradation. In cells expressing v-Src, Hakai-mediated ubiquitination might control the transport of E-cadherin to late endosomes and lysosomes for degradation (42). Given that Cdc42(T17N) is unable to dissolute adherens junctions, binding of Cdc42 to E-cadherin and Hakai may be important in directing ubiquitinated E-cadherin trafficking through endosomes to lysosomes for degradation. Together with its ability to regulate Hakai intracellular localization, activation of Cdc42 may ensure that ubiquitinated E-cadherin is targeted for lysosomal degradation. Constitutive activation of Cdc42 causes a enhanced rated of E-cadherin degradation.

Previously published research indicates that EGFR, Src, and Cdc42 positively regulate EMTs. Both EGFR and Src have been shown to regulate EMT by disrupting adherens junctions (43). The activation of Cdc42 has been suggested to be critical in preventing prosomitic mesenchymal cells from transitioning to epithelial cells (46). Mechanisms behind these effects have not been elucidated. Our data support the idea that increased signaling through Cdc42 to EGFR and Src contributes to a reduced epithelial cell polarity in breast cancer cells. Although Snail and TWIST likely control EMT by repressing E-cadherin expression (47), we propose that Cdc42, EGFR, and Src function as a signaling node during the early stages of EMT to initiate E-cadherin down-regulation by lysosomal degradation.

Implication for a Role of Cdc42 in Cancer Progression—E-cadherin has been extensively studied, and loss of function has been associated with cancer progression, invasion, and metastasis (48-50). Cdc42 has been implicated in regulation of cell migration, invasiveness, and metastasis (51). Overexpression of Cdc42 has been found in human breast cancers, and it is believed that the elevated expression results in enhanced signaling in tumor cells (17, 52, 53). Based on our studies, activation of Cdc42 may contribute to increased cancer invasion and metastasis by two complementary pathways, both of which are through the regulation of EGFR signaling. First, activation of Cdc42 inhibits c-Cbl-mediated ubiquitination and degradation of EGFR, resulting in sustained EGFR signaling (27). Second, this Cdc42-regulated increase in EGFR signaling likely contributes to the initial decrease in protein levels of E-cadherin at adherens junction by targeting E-cadherin for lysosomal degradation. The resulting destabilization of adherens junctions would then promote tumor progression toward more malignant states. The current studies establish a role for Cdc42 as a regulator of E-cadherin down-regulation and intracellular trafficking and further implicate Cdc42 as an important contributor to the invasive phenotype of human breast cancer cells.

	FOOTNOTES

 
^* 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.

¹ Both authors contributed equally to this work.

² An Interagency Oncology Task Force Fellow supported by the United States Food and Drug Administration and the NCI, National Institutes of Health, Bethesda, MD.

³ To whom correspondence should be addressed: Division of Monoclonal Antibodies, OBP/OPS/CDER/FDA, Bldg. 29B, Rm. 3NN-15, 29 Lincoln Dr., Bethesda, MD 20892-4555. Tel.: 301-827-0253; Fax: 301-827-0852; E-mail: wen.wu{at}fda.hhs.gov.

⁴ The abbreviations used are: EMT, epithelial to mesenchymal transitions; EGFR, epidermal growth factor (EGF) receptor; HA, hemagglutinin; DMEM, Dulbecco'smodifiedEagle'smedium;FBS,fetalbovineserum;siRNA,shortinterfering RNA; WCL, whole cell lysates; PBD, p21 binding domain; GST, glutathione S-transferase; wt, wild type; ERK, extracellular signal-regulated kinase.

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