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Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand

Abstract Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.


Introduction
The epidermal growth factor receptor (EGFR) family consisting of ErbB1 (EGFR), ErbB2, ErbB3 and ErbB4 is known to play a crucial part in the development and progression of cancers. The signal for cell survival, differentiation and proliferation is achieved through both homo-and heterodimerization of the receptors [1,2].
Anomalous increase in the expression of the ErbB2 is an identified factor for the development of several cancer types [1,3] and high ErbB2 levels parallel the worse prognosis for breast cancer patients [4][5][6]. ErbB2 also associates with ErbB3 in the hetero dimerization and is considered the most active dimer that initiates cancers [7,8]. ErbB2 is overexpressed in breast (30%) and ovarian (15-30%) cancer [9][10][11]. The myriad roles played by ErbB2 have made it one of the most preferred targets for the treatment of cancer. ErbB2 stringently internalizes efficiently recruiting back to the cell surface [12]. The mechanism of ErbB2 recycling and degradation is complex and appears to vary depending on the cell types. Various groups have shown that geldanamycin treatment efficiently internalizes the ErbB2 ligand [13][14][15]. Although the internalization of ErbB2 is enhanced when multivalently cross-linked with antibody such as anti-ErbB2 antibody sc-08 [13,[16][17][18][19], there is no known natural ligand that promotes the ErbB2 internalization till date. By phage display method, small peptides, which possess binding affinity to ErbB2, have been isolated [20][21][22]. EC-1 peptide, a cyclic 20-amino-acid-peptide, is also one of the artificial ErbB2 ligands isolated from random peptide library of phage display [23]. EC-1 peptide is considered to abolish the tyrosine phosphorylation of the intracellular domain of ErbB2 and to inhibit the proliferation of SK-BR-3 cells overexpressing ErbB2.
Previously, EC-1 peptide fused to GFP was shown to stimulate internalization of ErbB2 in SK-OV-3 cells whereas ErbB2 in SK-BR-3 cells retained on the surface [24]. Because anti-ErbB2 antibody sc-08 stimulated the internalization of ErbB2 in both cell lines, the mechanism of internalization stimulated by EC-1 peptide was considered quite different from that by anti-ErbB2 antibody sc-08. In this study, we designed multivalent form using EC-1 peptide fused to human IgG-Fc domain (EC-Fc) and bionanocapsule displaying ZZ-tag on the surface [25]. We then asked whether the multivalent forms stimulated the internalization of ErbB2 in SK-BR-3 cells.

Construction of expression plasmids
The expression plasmid for EC-Fc was constructed as follows. The DNA fragment coding human IgG-Fc domain was excised from the plasmid pBO593 [26] with restriction enzymes AgeIand NotIand then ligated at the 3Ј-end of a synthetic oligonucleotide coding for the EC-1 peptide. The resultant sequence coding EC-Fc was ligated to the 3Ј-end of the sequence coding the signal peptide derived from human RNase1 [27]. Finally, the sequence coding EC-Fc with a secretion signal peptide was cloned downstream of CMV promoter to construct the plasmid pBO854 with hygromycin resistant gene. Simultaneously, the expression vector for Fc domain without EC-1 moiety was constructed in the same procedure to construct the plasmid pBO853.

Preparation of EC-Fc protein
Chinese hamster ovary (CHO)

Competition EIA to estimate Kd value
The value of dissociation constant (Kd) between the ligand and ErbB2 was estimated as previously described with slight modification [24,28]

Preparation of multivalently displayed EC-1 peptide on bionanocapsule
To prepare the multivalent form of EC-1 peptide, EC-Fc proteins were displayed on the surface of bionanocapsule (BNC), which was composed of the recombinant surface antigen of hepatitis B virus [29]. To confer the high affinity for IgG Fc domain to BNC, the surface antigen was designed to fuse with the ZZ motif in the protein A derived from Staphylococcus aureus [25]

Biotinylation assay for the internalization of ErbB2
Biotinylation was performed as described previously [24]. Briefly, 5ϫ10 5

Analysis of cellular uptake of EC-Fc/BNC
The receptor-mediated cellular uptake of EC-Fc/BNC was evaluated. The cells in 60-mm dish were washed twice with ice-cold PBS and incubated with 2 nM (10 g/ml) of EC-Fc/BNC for 5 hrs at 4ЊC or 37ЊC. After incubation, the cells were washed twice with ice-cold PBS to remove unbound BNC and then collected by the treatment with 0.025% trypsin. After centrifugation at 6,000 rpm for 1 min the supernatant was discarded and the cell pellet was washed twice with ice-cold PBS. The cells were then lysed in the lysis buffer, further incubated for 20 min at 4ЊC and sonicated twice. The cell extracts were clarified by centrifugation for 5 min at 4ЊC. Twenty microlitres of anti-HBsAg microbeads suspension was added to the extracts and incubated overnight at 4ЊC. After centrifugations for 30 sec at 4ЊC, the beads were washed three times in PBS, suspended in Laemmli buffer supplemented with ␤-mercaptoethanol, heated for 5 min at 95ЊC, and subjected to SDS-PAGE followed by Western blotting.

Western blotting and image analysis
Proteins resolved on acrylamide gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, VA). The membrane was blocked with 10% skim milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl containing 0.1% Tween-20 (TBST). The blots were then probed with a biotinylated polyclonal anti-human IgG antibody diluted to 1:1,000 (Dako, Denmark) or anti-preS-1 monoclonal antibody (Beacle, Japan) conjugated to HRP diluted to 1:15,000 in blocking buffer, followed by a HRP-Avidin (Zymed, CA) in TBST containing 10% skim milk. The HRP signal was developed with Western Lighting™ and Western Lighting Plus Chemiluminescence Reagent (Perkin Elmer, MA) and the intensity of the bands were visualized by LAS image 4000 (Fujifilm, Japan). Quantitative assessments of the relative intensity of the blots were performed with NIH ImageJ. Figure 1B (Fig. 1C). The majority of EC-Fc was considered divalent form of EC-1 peptide even if the oligomeric forms such as hexamer and higher oligomer were formed because high oligomerization should precipitate EC-Fc and prevent it from working as ligand of ErbB2. For the susceptibility to the reducing conditions, EC-Fc was treated with DTT at various concentrations ranging from 0.5 to 5 mM at 4ЊC and 25ЊC (Fig. 1D). As the result, no order of disulfide bonds appeared to be present in the sensitivity to the reduction reagent in oligomeric forms because the reduction did not stop at a dimer even in low concentration of DTT. The specific binding affinity of EC-Fc to sErbB2 was evaluated by competition EIA (Fig. 1E). Scatchard plot revealed a high affinity binding site with a Kd of 26 nM, whereas a low affinity site was estimated to be 360 M.

Internalization of ErbB2 was induced by EC-Fc in SK-BR-3 cells
Because the specific binding of EC-Fc to ErbB2 was confirmed, we then checked uptake of ErbB2 upon incubation with EC-Fc. For this assay, surface biotinylated cells were prepared and used. After incubation with EC-Fc, the biotin-labelled molecules retained on the cell surface were stripped off by the reducing reagent, whereas the molecules internalized were kept labelled with biotin so that they should be immunoprecipitated for the further analysis. The anti-ErbB2 antibody sc-08, which is known to induce internalization of ErbB2 in either SK-OV-3 cells or SK-BR-3 cells [13,34], was used as a positive control. When evaluated on SK-BR-3 cells, EC-Fc was found to induce the internalization of ErbB2 ( Fig. 2A). This internalization was estimated approximately 50% of that induced by anti-ErbB2 antibody sc-08. Internalized ErbB2 was observed when SK-OV-3 cells were treated with EC-Fc (Fig. 2B). In SK-OV-3 cells, the internalized ErbB2 induced by EC-Fc was almost equivalent with that induced by antibody sc-08. EC-Fc at 1 M showed internalization of ErbB2 equivalent to that induced by anti-ErbB2 antibody sc-08 at 6.67 nM. This internalization was dependent on the doses of EC-Fc in a similar manner of monovalent EC-1 peptide fused to eGFP (EC-eGFP) [24]. When the biotinylated cells were treated with 5 M EC-Fc, the internalized fraction of ErbB2 was increased up to three folds compared to that of the untreated cells at 37ЊC (Fig. 2C)

Internalization of ErbB2 was enhanced by multivalent form of EC-Fc in SK-BR-3 cells
The multivalent form of EC-1 peptide was prepared exploiting the affinity of ZZ-BNC for IgG Fc region (Fig. 3A) Figure 3B, the soluble Fc/BNC was reduced to 30% at a molar ratio of ZZ-BNC to Fc protein at 1:100 when judged from the fluorescence in the supernatant. Similarly, when human IgG was used, the soluble IgG/BNC was found reduced to 10% at a molar ratio of ZZ-BNC to IgG protein at 1:100. The amount of Fc protein bound to ZZ-BNC in the supernatant was further estimated from intensity of the band detected by Western blotting (Fig. 3C) ErbB2 internalization mediated by EC-Fc/BNC was evaluated by the cellular uptake of EC-Fc/BNC (Fig. 3D-F). The SK-BR-3 cells were incubated with either EC-Fc/BNC or Fc/BNC for 5 hrs at 37ЊC or 4ЊC (Fig. 3D). As a result, EC-Fc/BNC showed significant cellular uptake at 37ЊC in SK-BR-3 cells. The EC-Fc/BNC cellular uptake into SK-BR-3 cells was found not only dose dependent in the range from 1 to 10 nM (Fig. 3E) and time course dependent up to 8 hrs at 2 nM (Fig. 3F). These results suggest that multivalent EC-1 peptide displayed on BNC might make its internalization into the cells efficient. The time course changes in the internalization of BNC in SK-BR-3 cells were evaluated by incubating cells with EC-Fc/BNC for various time periods under a confocal microscope (Fig. 3G). To demonstrate that observed localization or internalization of the receptor is not an artefact of fixation and cell permeabilization, we assessed the internalization of the EC-Fc/BNC with live cell imaging (Fig. S1)

Comparative study of the effect by EC-Fc and EC-Fc/BNC
The specific binding of EC-1 ligand to cells overexpressing ErbB2 was assessed by immunostaining (Fig. 4A) (Fig. 4C).

EC-Fc/BNC internalized through GEEC pathway in SK-BR-3 cells
Cells adopt divergent pathways for the endocytosis of the cargos and receptors. The key pathways were categorized into clathrin-dependent and clathrin-independent mechanisms [33]. The clathrin-independent pathway is further classified into caveolar and GPI-anchored early endocytic compartments (GEEC) pathways [33,34]. ErbB2 is thought to be internalizing in SK-OV-3 through clathrin-mediated internalization [24]. In this study we found that the mechanism of internalization of ErbB2 present in SK-OV-3 cells is deficient in SK-BR-3 cells. We tried to identify the difference between the two cell lines using DNA microarray (Fig. S2) -Cav1 antibody (Fig. 5A). SK-BR-3 did not show the presence of caveolin-1 suggesting that the impaired caveolar mechanism prevailing in the cell, which was consistent with the finding by the previous reports [13,35]

. Because caveolin-1 was detected in SK-OV-3 cells the internalization pathway deficient in SK-BR-3 cells might be attributed to caveolae. The mechanistic approach for the uptake of the EC-Fc/BNC was assessed in SK-BR-3 cells using inhibitors for other pathways. SK-BR-3 cells were treated with EC-Fc/BNC in the presence or absence of inhibitors for clathrin, and GEEC pathway and the internalization was assessed.
When the cells were treated with 100 nM CPZ, an amphiphilic drug, which inhibits the clathrinmediated pathway, the internalization of the EC-Fc/BNC was unaffected (Fig. 5B) [36,37]. The colocalization of EC-Fc/BNC with EEA-1 was assessed with anti-EEA-1 antibody under confocal microscope at various time periods starting from 5, 30 and 90 min and the colocalization was found to be maximum at 30 min (Fig. S3).

Most of the cargos irrespective of their route merges with Rab5 and early endosome antigen-1 (EEA-1) enriched in early endosomes, which is further sorted into various intracellular destinations
When the cells were treated with 5 mM m␤CD, which dislodges the cholesterol from the surface and inhibit the cdc42 activity, for 30 min the internalization was blocked and the cells untreated showed considerable uptake of EC-Fc/BNC. The bound fractions of EC-Fc/BNC were treated with and without acid stripping to ensure the colocalized fraction inside the cells (Fig. 5C). The colocalization was calculated to more than 60%, which was reduced in the internalization of EC-Fc/BNC with the m␤CD treatment. To assess the surface binding of the EC-Fc/BNC after the inhibitor treatment the cells were stained for the ErbB2 and EC-Fc/BNC at various concentration of the m␤CD (Fig. S4). The inhibitor treatment did not deteriorate the binding affinity of the EC-Fc/BNC to ErbB2. These results were further confirmed with flow cytometric studies (Fig. S5).

Discussion
The EC-Fc was designed as dimer due to the hinge region in IgG Fc domain (Fig. 1A). However, we found higher oligomeric forms such as tetramer and hexamer. These higher order forms were supposed to be divalent as described in 'Results' and Figure 1C.

hr at 37ЊC in MCF-7 cells, SK-BR-3 cells, and SK-OV-3 cells. MCF-7 cells were used as the control for low ErbB2 expression. The cells were stained with anti-human IgG labelled with FITC. Bars depict 10 m. The treated cells were also subjected to flow cytometoric analysis for the surface bound fraction of EC-Fc and Fc ligand with and without BNC in MCF-7, SK-BR-3 and SK-OV-3, respectively. Untreated cells were shown in shadow (grey). Treatment with EC-Fc ligand Fc ligand, EC-Fc/BNC and Fc/BNC was marked as green, magenta, blue and orange, respectively. (B) SK-BR-3 cells were treated with different concentration of EC-Fc or Ec-Fc/BNC. The cell surface receptors were reversibly biotinylated with NHS-SS-Biotin and were incubated with EC-Fc, Fc, EC-Fc/BNC or Fc/BNC for 2 hrs at 37ЊC. SK-BR-3 cells left untreated at 4ЊC for 2 hrs were shown as lane '0'. '0*' corresponds to the cells treated with Fc ligand (top) and Fc/BNC (lower) at a concentration of 200 nM, respectively. After the treatments, cells were lysed, immunoprecipitated with avidin agarose and were subjected to Western blot to detect
ErbB2 with anti-ErbB2 antibody. sc-08 antibody treatment was taken as positive control for endocytosis of ErbB2. Transferrin receptor (TfR) was monitored as an internal control for the experiments. (C) The bands from the Western blots were densitometrically analysed with ImageJ and the intensity each band was normalized by the intensity when treated with antibody sc-08. monovalent form of EC-1 peptide did not induce internalization [24]. The internalization of ErbB2 was found dependent on the concentration in the range of 20-200 nM for EC-Fc and appeared to be enhanced when 20-100 nM EC-Fc was displayed on BNC (Fig. 4B) (Fig. 3D-F). It is thought that the processing and degradation of the ErbB2 is different in various cell lines [24,34,35]. The CPZ at 100 nM, which inhibits the formation of clathrin-coated pits, inhibited the internalization of ErbB2 by EC-1 peptide fused to eGFP in SK-OV-3 [24]. On the contrary, the same concentration of CPZ did not block the internalization of ErbB2 in SK-BR-3 cells. In this study, we confirmed the absence of caveolin-1 in SK-BR-3 cells using microarray and immunostaining where it showed around 300-fold increment in the expression of Cav-1 in SK-OV-3 cells (Fig. S1). Recent studies support our observation that the SK-BR-3 cells lack caveolae [13,35]. ErbB2 internalization after geldanamycin treatment showed GEEC or clathrin-independent compartments (CLIC) pathway, which ultimately fuses with the classical pathway for the degradation in lysosomes, is independent of caveolar and clathrin pathway [35].
In this study we employed m␤CD, a cholesterol dislodging oligosaccharide, for deciphering the mechanism behind the internalization of the EC-Fc/BNC in SK-BR-3 cells, as an inhibitor for the blockade of GEEC pathway. Cholesterol depletion with m␤CD inhibits both caveolar [38] and GEEC pathway [39,40]. Earlier it was shown that cholesterol level determines the internalization of GPIanchored proteins and with clostridium difficile toxin B [34,39]. Because SK-BR-3 cells lack caveolar machinery, the blockade with m␤CD will only correspond its effect in blocking the internalization of GEEC-mediated mechanism. The internalization of ErbB2 is sensitive to the depletion of cholesterol, which is essential for most of the clathrin-independent pathways. GEECs are formed as compartments free of clathrin, caveolin and dynamin, which are short lived (2-5 min) and ultimately fuses with EEA-1 enriched early endosome [33,38]. It was shown that cdc42 is responsible for the dynamindependent and clathrin-independent internalization of GPI-anchored proteins. It was also reported earlier that cdc42 activation recruits actin polymerization machinery rendering this pathway sensitive to inhibitors of cholesterol depletion and actin polymerization [39]. This pathway specifies the visible difference in the uptake of the EC-Fc/BNC between SK-OV-3 and SK-BR-3 cells.
Another important molecule, which plays a key role in the GEEC pathway, is EEA-1 that contains an FYVE finger, which interacts with PI3K [41,42]. PI3K phosphorylates Rab 5 that helps the EEA-1 to localize to early endocytic compartments [36]. The colocalization of EC-Fc/BNC with EEA-1, which corresponds the preliminary step in the endosomal pathways before transferring it to the sorting endosomes, was observed (Fig. S2). Because both caveolar and clathrin pathways are impaired in SK-BR-3 cells as described earlier, the colocalization should correspond to GEEC pathway.
In this study, we revealed the presence of GEEC pathway as ErbB2 internalization, which critically differs between SK-BR-3 cells and SK-OV-3 cells using EC-1 peptide in various forms. As for the nanoparticle internalization, the pathways of clathrin and caveolar-independent endocytosis have not been described until now [43]. The internalization of the carrier particle was described repeatedly on folate receptor (FR) tagged nanoparticles [44]. In these reports, mechanism of the carrier internalization emphasizes the FR internalization through GEEC pathway. Similarly, EC-Fc should utilize the mechanism of internalization of ErbB2 in this study even when they are displayed on BNC (Fig. 4B).
It is considered that ZZ-BNC is an efficient nano-machine for molecular targeting in drug delivery system displaying antibodies on the surface [25,29,45] (Fig. S5A). The histograms showed overlapping of the peak tops indicating that the surface binding is majorly unaffected by the m␤CD treatment. The peak (blue), which corresponds to approximately 20% of the total cell counts, orienting towards the untreated cells (grey peak) indicates the presence of a different population of cells that are incapable of binding to EC-Fc/BNC after m␤CD treatment. Simultaneously, another set cells were treated with EC-Fc/BNC labeled with FITC in the presence or absence of 5 mM of m␤CD at 37°C for 30 minutes. Cells were then trypsinized to remove the surface fraction, fixed and permeabilized, and the internalized fraction was analyzed adopting the same procedure with FACS (Fig. S5B, C). m␤CD treated SK-BR-3 cells showed a peak point in the histogram which overlaid the peak area of untreated cells indicating the m␤CD inhibits the internalization, while the cells without m␤CD treatment drifted from the untreated cells towards the right. Total intensity of the internalized fraction was assessed from the FACS data were plotted on to a graph (Fig. S5). m␤CD suppressed the internalization to approximately 50% when compared to the absence of m␤CD treatment. M: m␤CD; T: trypsin. Error bars indicate standard error.
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