A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2–HER3 heterodimers, and little is known regarding EGFR–HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR–HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR–HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR–HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR–HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR–HER2 heterodimer. Disruption of the lipid raft with MβCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MβCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR–HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR–HER2 heterodimer.

in the dark for 30 min and DNA contents were measured with BD FACSCanto™ II. Finally, the results were analyzed with FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA)

Cell Lysates and Immunoblotting
Cell lysates were prepared as previously described [51]. Briefly, the cells were collected by scraping and lysedin ice-cold Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) containing a protease and phosphatase inhibitor cocktail 0.02% NaN3, 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 µM pepstatin A, 10 µg/mL aprotinin, 0.5 mM Na 3 VO 4 ,). Following the incubation on ice for 1 h, the cell lysates were centrifuged at 21,000× g for 15 min at 4 • C. The supernatant was collected, mixed with equal volume of 2× sample buffer, and boiled for 5 min. Following the gel electrophoresis, the proteins were transferred to the nitrocellulose membrane. The nitrocellulose membrane was immunoblotted with various primary antibodies as indicated. The protein bands were detected and analyzed by using Odyssey CLx imaging system (LI-COR biotechnology Inc., Lincoln, NE, USA).

Immunofluorescence
Cells (10 5 ) were seeded on 15 mm round cover glass in 24 well-plates, and were cultured for 48 h to allow the cells to settle and attach. Cells were treated in the same way as described in 5.1. Following the treatment, cells were washed with ice-cold PBS and then fixed with −20 • C methanol for 5 min. Afterwards, the cells were washed with TBS and blocked with TBS containing 1% bovine serum albumin (BSA) for 60 min. Cell were incubated in 2 µg/mL of primary antibodies, as indicated, for 60 min. The cells were washed and then incubated in 1 µg/mL FITC-conjugated and/or 1 µg/mL rhodamine-conjugated secondary antibodies for 60 min in the dark. Afterwards, the cells were washed with TBS and then incubated in 1 µg/mL DAPI solution for 5 min. The coverslips were mounted on microscope slides and were examined with a Deconvolution Microscope system (GE Healthcare Life Science, Saskatoon, SK, Canada). All of the images were deconvolved. The selected images were line scanned with the software SoftWoRx embedded in the Microscope system. The line scan measured the intrinsic intensity of the individual fluorescence channel, and was not affected by changing the contrast and brightness.

Lipid Rafts Associated Proteins Isolation
The proteins present in lipid rafts were isolated from BT474 cells by Bio-Rad ReadyPrep™ Protein Extraction Kit (Signal) kit according to the instruction provided by manufacturer. Due to the presence of a high amount of cholesterol and sphingolipids in the lipid rafts, these regions of the cell membrane are insoluble in nonionic detergents. Therefore, treating the cells with nonionic detergents, provided by the kit, will solubilize hydrophilic regions of the cells, and leave the lipid raft microdomains and their associated proteins insoluble. This insoluble fraction can be solubilized in a specific buffer named protein solubilization buffer (PSB) provided by the company.

Statistical Analysis
Blot band intensity was quantified by ImageJ software and normalized to total expression of protein of interest. Data were statistically analyzed by one-way analysis of variance (ANOVA) using Prism software (GraphPad Software, La Jolla, CA, USA). Data were presented as mean and standard deviation. p < 0.05 was considered as statistically significant.

The Effect of Trastuzumab on the Proliferation and Cell Cycle Progression of HER2-Positive Breast Cancer Cells
We first determined if trastuzumab inhibits the proliferation of BT474 and SKBR3 cells. For this purpose, the MTT cell viability kit was used to assess cell proliferation. The cells were treated with trastuzumab for 72 h under three different conditions including: (1) DMEM only, without adding any supplements, (2) DMEM supplemented with 10% FBS, and (3) DMEM supplemented with EGF. It is worth mentioning that even though culturing the cells in the medium without FBS slowed the cell growth, it did not have adverse effects on the health of the cells. Our results showed that trastuzumab inhibited cell proliferation in the absence and the presence of 10% FBS and EGF in both BT474 and SKBR3 cells. As a positive control, we treated the cells with HER2-specific small molecule tyrosine kinase inhibitor, CP-724714, which inhibited the proliferation of BT474 and SKBR3 cells under all conditions ( Figure 1).
Blot band intensity was quantified by ImageJ software and normalized to total expression of protein of interest. Data were statistically analyzed by one-way analysis of variance (ANOVA) using Prism software (GraphPad Software, La Jolla, CA, USA). Data were presented as mean and standard deviation. p < 0.05 was considered as statistically significant.

The Effect of Trastuzumab on the Proliferation and Cell Cycle Progression of HER2-Positive Breast Cancer Cells
We first determined if trastuzumab inhibits the proliferation of BT474 and SKBR3 cells. For this purpose, the MTT cell viability kit was used to assess cell proliferation. The cells were treated with trastuzumab for 72 h under three different conditions including: (1) DMEM only, without adding any supplements, (2) DMEM supplemented with 10% FBS, and (3) DMEM supplemented with EGF. It is worth mentioning that even though culturing the cells in the medium without FBS slowed the cell growth, it did not have adverse effects on the health of the cells. Our results showed that trastuzumab inhibited cell proliferation in the absence and the presence of 10% FBS and EGF in both BT474 and SKBR3 cells. As a positive control, we treated the cells with HER2-specific small molecule tyrosine kinase inhibitor, CP-724714, which inhibited the proliferation of BT474 and SKBR3 cells under all conditions ( Figure 1). Studies have revealed that trastuzumab inhibits the proliferation of HER2-positive breast cancer cells, likely through the inhibition of cell cycle progression [67][68][69][79][80][81]. To assess if this is the case for our observed inhibition in Figure 1, we examined if trastuzumab inhibits the cell cycle progression in BT474 and SKBR3 cells by flow cytometry. We treated the cells with trastuzumab at 10 µ g/mL concentration in the presence and absence of 10% FBS for 72 h. We showed that trastuzumab treatment significantly increased the population of cells in G1 phase under all conditions in both BT474 and SKBR3 cells ( Figure 2). As a positive control, CP-724714 also increased cell population in G1 phase Studies have revealed that trastuzumab inhibits the proliferation of HER2-positive breast cancer cells, likely through the inhibition of cell cycle progression [67][68][69][79][80][81]. To assess if this is the case for our observed inhibition in Figure 1, we examined if trastuzumab inhibits the cell cycle progression in BT474 and SKBR3 cells by flow cytometry. We treated the cells with trastuzumab at 10 µg/mL concentration in the presence and absence of 10% FBS for 72 h. We showed that trastuzumab treatment significantly increased the population of cells in G1 phase under all conditions in both BT474 and SKBR3 cells ( Figure 2). As a positive control, CP-724714 also increased cell population in G1 phase similarly to trastuzumab ( Figure 2). These data suggest that trastuzumab blocks cell proliferation by arresting cells in G1 phase.

R REVIEW
6 of similarly to trastuzumab ( Figure 2). These data suggest that trastuzumab blocks cell p liferation by arresting cells in G1 phase.

The Effect of Trastuzumab on EGF-Induced Formation of EGFR-HER2 Heterodimers
We next examined the effects of trastuzumab on EGF-induced formation of EGF HER2 heterodimers by co-immunoprecipitation (co-IP) assay in BT474 and SKBR3 ce For this purpose, HER2 antibody was used as a primary antibody to precipitate HE receptors, and heterodimer formation was identified by immunoblotting of EGFR. W showed that HER2 formed a heterodimer with EGFR, with or without EGF stimulatio Quantification of the data showed that addition of EGF stimulated the dimerization b tween EGFR and HER2. Interestingly, trastuzumab did not block the heterodimerizati (p > 0.1) between HER2 and EGFR with or without EGF stimulation in both BT474 a

The Effect of Trastuzumab on EGF-Induced Formation of EGFR-HER2 Heterodimers
We next examined the effects of trastuzumab on EGF-induced formation of EGFR-HER2 heterodimers by co-immunoprecipitation (co-IP) assay in BT474 and SKBR3 cells. For this purpose, HER2 antibody was used as a primary antibody to precipitate HER2 receptors, and heterodimer formation was identified by immunoblotting of EGFR. We showed that HER2 formed a heterodimer with EGFR, with or without EGF stimulation. Quantification of the data showed that addition of EGF stimulated the dimerization between EGFR and HER2. Interestingly, trastuzumab did not block the heterodimerization (p > 0.1) between HER2 and EGFR with or without EGF stimulation in both BT474 and SKBR3 cells ( Figure 3). HER2-EGFR and HER2-HER3 heterodimer formation was assesse in the presence and absence of EGF and HRGα at 50 ng/mL, respectively. The expression of HE receptors in both IP and total cell lysate (TCL) samples was revealed by immunoblotting. HER receptors were precipitated using HER2-specific antibody as primary antibody, followed by im munoblotting with the indicated antibodies. Cells treated with normal human IgG were used a negative control. **: p < 0.01,

The Effect of Trastuzumab on the Phosphorylation of EGFR and HER2
To determine the effect of trastuzumab on the phosphorylation of EGFR and HER we treated the BT474 and SKBR3 cells with trastuzumab (10 µ g/mL) in the presence o absence of EGF for 1 h. The cells were also treated with IgG (10 µ g/mL) as the negativ control and CP-714724 (1 µ M) as the positive control ( Figure 4). We showed that EG stimulated EGFR and HER2 phosphorylation in both cell lines, as expected. Interestingly trastuzumab did not inhibit, but actually stimulated EGF-induced phosphorylation o EGFR and HER2 in BT474 cells. For SKBR3 cells, EGF did not inhibit or stimulate EGF and HER2 phosphorylation ( Figure 4). HER2-EGFR and HER2-HER3 heterodimer formation was assessed in the presence and absence of EGF and HRGα at 50 ng/mL, respectively. The expression of HER receptors in both IP and total cell lysate (TCL) samples was revealed by immunoblotting. HER2 receptors were precipitated using HER2-specific antibody as primary antibody, followed by immunoblotting with the indicated antibodies. Cells treated with normal human IgG were used as negative control. **: p < 0.01.

The Effect of Trastuzumab on the Phosphorylation of EGFR and HER2
To determine the effect of trastuzumab on the phosphorylation of EGFR and HER2, we treated the BT474 and SKBR3 cells with trastuzumab (10 µg/mL) in the presence or absence of EGF for 1 h. The cells were also treated with IgG (10 µg/mL) as the negative control and CP-714724 (1 µM) as the positive control ( Figure 4). We showed that EGF stimulated EGFR and HER2 phosphorylation in both cell lines, as expected. Interestingly, trastuzumab did not inhibit, but actually stimulated EGF-induced phosphorylation of EGFR and HER2 in BT474 cells. For SKBR3 cells, EGF did not inhibit or stimulate EGFR and HER2 phosphorylation ( Figure 4).
We further examined the effect of trastuzumab on the phosphorylation of EGFR and HER2 by immunofluorescence. Both BT474 and SKBR3 cells were treated with IgG (control) or trastuzumab for 1 h, and then stimulated with EGF. As shown in Figure 5A, in the absence of EGF, EGFR was slightly phosphorylated, but HER2 maintained a higher level of phosphorylation. GF stimulated strong phosphorylation of both EGFR and HER2. Treatment with trastuzumab did not show any observable inhibitory effects on the phosphorylation of EGFR and HER2. Following EGF stimulation for 30 min, phosphorylated EGFR was mostly internalized to the endosomes, but phosphorylated HER2 mostly stayed in the plasma membrane ( Figure 5B). We further examined the effect of trastuzumab on the phosphorylation of EGFR and HER2 by immunofluorescence. Both BT474 and SKBR3 cells were treated with IgG (control) or trastuzumab for 1 h, and then stimulated with EGF. As shown in Figure 5A, in the absence of EGF, EGFR was slightly phosphorylated, but HER2 maintained a higher level of phosphorylation. GF stimulated strong phosphorylation of both EGFR and HER2. Treatment with trastuzumab did not show any observable inhibitory effects on the phosphorylation of EGFR and HER2. Following EGF stimulation for 30 min, phosphorylated EGFR was mostly internalized to the endosomes, but phosphorylated HER2 mostly stayed in the plasma membrane ( Figure 5B). It has been shown that HER2 contains multiple phosphotyrosine (pY) residues that may be differentially regulated [51,58]. In the above experiments, we have been using antibody against HER2 pY1139 to determine the phosphorylation status of HER2. Here, we further examined the phosphorylation status of other HER2 pY residues including pY1005, pY1112, pY1127, pY1196, and pY1248 ( Figure 6). We showed that EGF stimulated the phosphorylation of all the HER2 pY residues and trastuzumab did not have observable effects on the phosphorylation of these HER2 pY residues.
Together, our data indicated that EGF induced HER2 phosphorylation in both BT474 and SKBR3 cells. Trastuzumab did not inhibit HER2 phosphorylation at all major pY residues, with or without EGF stimulation. It has been shown that HER2 contains multiple phosphotyrosine (pY) residues that may be differentially regulated [51,58]. In the above experiments, we have been using antibody against HER2 pY1139 to determine the phosphorylation status of HER2. Here, we further examined the phosphorylation status of other HER2 pY residues including pY1005, pY1112, pY1127, pY1196, and pY1248 ( Figure 6). We showed that EGF stimulated the phosphorylation of all the HER2 pY residues and trastuzumab did not have observable effects on the phosphorylation of these HER2 pY residues. Figure 5. The effects of trastuzumab on EGF-induced phosphorylation of EGFR and HER2 in BT474 and SKBR3 cells. Cells were incubated with TRZ (10 µg/mL) or human IgG for 1 h and then stimulated with EGF (50 ng/mL). (A) Following EGF stimulation for 15 min, the phosphorylation of EGFR and HER2 (green) was determined by antibody to phosphorylated EGFR (pEGFR, pY1086) and pHER2 (pY1139), respectively. The localization of EGFR and HER2 (red) was determined by antibodies to by total EGFR and HER2, respectively. Yellow indicates the co-localization. the size bar = 20 µm. (B) Localization of pEGFR (pY1086) and pHER2 (pY1139) following EGF stimulation for 30 min. the size bar = 20 µm.

The Effect of Trastuzumab on HER2-Mediated Downstream Signaling Pathways
We next examined if trastuzumab exerted any inhibitory effects on HER2-mediated downstream signaling. The AKT pathway and ERK pathway are the two most studied and important signaling pathways downstream; EGFR and HER2 and were examined in this study. Both BT474 and SKBR3 cells were treated with trastuzumab (10 µg/mL) for 1 h and then stimulated with EGF (50 ng/mL) for 30 min. Similarly, treatment with IgG (10 µg/mL) and CP-714724 (1 µM) were used as negative control and positive control, respectively. The activation of AKT and ERK were determined by examining their phosphorylation status through immunoblotting. It is well established that the full activation of AKT requires the phosphorylation of both T308 and S473. We examined the effects of trastuzumab on the AKT T308 and S473 phosphorylation with specific antibodies, and showed that trastuzumab inhibited the AKT phosphorylation at both T308 and S473 phosphorylation sites, with or without EGF stimulation in both BT474 and SKBR3 cells (Figure 7). However, the effects of trastuzumab on the phosphorylation of ERK are weaker. Trastuzumab did not inhibit the basal ERK phosphorylation in both BT474 and SKBR3 cells. While it inhibited EGF-induced phosphorylation of ERK in SKBR3 cells, trastuzumab only slightly inhibited EGF-induced ERK phosphorylation in BT474 cells, which is statistically insignificant (Figure 7). Together, these results indicated that trastuzumab strongly inhibited EGF-induced Akt c.
Cells 2022, 11, x FOR PEER REVIEW 10 of 20 Figure 6. The effects of trastuzumab on EGF-induced phosphorylation of HER2 at various pY sites including pY1005, pY1112, pY1127, pY1196, and pY1248 in BT474 and SKBR3 cells. Cells were incubated with TRZ (10 µ g/mL) or human IgG for 1 h and then stimulated with EGF (50 ng/mL) for 15 min. The phosphorylation of various HER2 pY sites (green) were determined by specific antibodies followed by FITC conjugated secondary antibody, and total HER2 (red) was revealed by anti-HER2 antibody followed by TRITC conjugated secondary antibody. Yellow indicates the co-localization. the size bar = 20 µ m.
Together, our data indicated that EGF induced HER2 phosphorylation in both BT474 and SKBR3 cells. Trastuzumab did not inhibit HER2 phosphorylation at all major pY residues, with or without EGF stimulation.

The Effect of Trastuzumab on HER2-Mediated Downstream Signaling Pathways
We next examined if trastuzumab exerted any inhibitory effects on HER2-mediated downstream signaling. The AKT pathway and ERK pathway are the two most studied and important signaling pathways downstream; EGFR and HER2 and were examined in this study. Both BT474 and SKBR3 cells were treated with trastuzumab (10 µ g/mL) for 1 h and then stimulated with EGF (50 ng/mL) for 30 min. Similarly, treatment with IgG (10 µg/mL) and CP-714724 (1 µ M) were used as negative control and positive control, respectively. The activation of AKT and ERK were determined by examining their phosphorylation status through immunoblotting. It is well established that the full activation of AKT requires the phosphorylation of both T308 and S473. We examined the effects of trastuzumab on the AKT T308 and S473 phosphorylation with specific antibodies, and showed that trastuzumab inhibited the AKT phosphorylation at both T308 and S473 phosphorylation sites, with or without EGF stimulation in both BT474 and SKBR3 cells ( Figure  7). However, the effects of trastuzumab on the phosphorylation of ERK are weaker. Trastuzumab did not inhibit the basal ERK phosphorylation in both BT474 and SKBR3 cells. While it inhibited EGF-induced phosphorylation of ERK in SKBR3 cells, trastuzumab only slightly inhibited EGF-induced ERK phosphorylation in BT474 cells, which is statistically insignificant (Figure 7). Together, these results indicated that trastuzumab strongly inhibited EGF-induced Akt c. Figure 6. The effects of trastuzumab on EGF-induced phosphorylation of HER2 at various pY sites including pY1005, pY1112, pY1127, pY1196, and pY1248 in BT474 and SKBR3 cells. Cells were incubated with TRZ (10 µg/mL) or human IgG for 1 h and then stimulated with EGF (50 ng/mL) for 15 min. The phosphorylation of various HER2 pY sites (green) were determined by specific antibodies followed by FITC conjugated secondary antibody, and total HER2 (red) was revealed by anti-HER2 antibody followed by TRITC conjugated secondary antibody. Yellow indicates the co-localization. the size bar = 20 µm.

The Effect of Trastuzumab on Lipid Raft Localization and Function of EGFR-HER2 Heterodimer
The above research revealed interesting effects of trastuzumab. In HER2-positive BT474 and SKBR3 cells, trastuzumab did not inhibit the dimerization of EGFR-HER2 receptors, and the phosphorylation of EGFR and HER2. However, trastuzumab strongly inhibited EGF-induced phosphorylation of AKT. Moreover, trastuzumab blocked proliferation of both BT474 and SKBR3 cells by arresting the cells in G1 phase. These results suggest that trastuzumab inhibits EGF-induced activation of AKT and cell cycle progression/proliferation by a mechanism other than the dimerization and phosphorylation of EGFR and HER2.
Previous studies have shown that lipid raft localization of HER receptors, including EGFR and HER2, plays a critical role in the activation of downstream signaling pathways [22,[82][83][84][85][86][87]. In addition, trastuzumab binds to the juxta-membrane region of HER2 [88], which likely affects the movement of the HER2 transmembrane domain within the plasma membrane. Indeed, it has been shown that trastuzumab can affect HER2 receptors' localization and arrangement in the membrane of breast cancer cells [89,90]. Therefore, we proposed that trastuzumab inhibits the lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt activation.
To test this hypothesis, we first investigated the possible effect of trastuzumab on HER2 and EGFR localization to the lipid raft microdomains of the cell membrane. We treated the cells with trastuzumab (10 µg/mL) with or without EGF for 1 h, and isolated the proteins associated with lipid rafts using Bio-Rad ReadyPrep™ Protein Extraction Kit (Signal). The cells treated with IgG were used as the negative control. The level of HER2 and EGFR in isolated lipid rafts were examined by immunoblotting. We showed that EGF stimulated the translocation of EGFR and HER2 to the lipid raft. Moreover, the localization of HER2 and EGFR to the lipid rafts was decreased after the treatment of cells with trastuzumab in both BT474 and SKBR3 cells ( Figure 8A).
We further studied the effects of trastuzumab on the lipid raft localization of EGFR and HER2 by indirect immunofluorescence. Alexa Fluor 488 conjugated cholera toxin subunit (CT-B), which binds to the raft constituent ganglioside GM1, was used as the marker for lipid rafts. The co-localization of EGFR/HER2 and CTB with or without EGF stimulation were examined by fluorescence microscopy. As shown in Figure 8B, the peaks of green indicated the location of lipid rafts along the PM (line scan). In the absence of EGF stimulation, both EGFR and HER2 (red) distributed smoothly along the PM, not concentrated in the lipid rafts. Following EGF stimulation for 30 min, the intensity peak of HER2 and EGFR (red) overlapped with the peak of CT-B (green) as shown in line scan, indicating the translocation of EGFR and HER2 to lipid rafts. However, pretreatment with trastuzumab blocked the lipid translocation of both HER2 and EGFR. Both HER2 and EGFR showed smooth distribution along the PM, and were not co-peaked with CT-B as shown in the line scan.
Together, our data indicated that trastuzumab strongly blocked EGF-induced translocation of pHER2 and pEGFR to the lipid rafts (Figure 8).
the proteins associated with lipid rafts using Bio-Rad ReadyPrep™ Protein Extraction Kit (Signal). The cells treated with IgG were used as the negative control. The level of HER2 and EGFR in isolated lipid rafts were examined by immunoblotting. We showed that EGF stimulated the translocation of EGFR and HER2 to the lipid raft. Moreover, the localization of HER2 and EGFR to the lipid rafts was decreased after the treatment of cells with trastuzumab in both BT474 and SKBR3 cells ( Figure 8A). Figure 8. The effect of trastuzumab on EGF-induced lipid raft localization of EGFR-HER2 heterodimer in BT474 cells. (A) EGFR and HER2 level in isolated lipid rafts. The cells were incubated with trastuzumab or normal human IgG (10 µg/mL) for 1 h and then stimulated with EGF (50 ng/mL) for 30 min. Proteins associated with lipid rafts were isolated by using Bio-Rad ReadyPrep™ Protein Extraction Kit (Signal). The level of HER2 and EGFR receptors in isolated lipid rafts were examined by immunoblotting. Each value is the average of three experiments and the error bar is the standard error. *: p < 0.05, **: p < 0.01. (B) Co-localization (yellow) of pEGFR (red) and pHER2 (red) with Alex Fluor 488 conjugated CT-B (green). Cells were incubated with CB-T (10 µg/mL) with or without trastuzumab (10 µg/mL) for 1 h and then stimulated with EGF for 30 min. The localization of EGFR or HER2 was revealed by TRITC conjugated secondary antibody following the incubation with the primary antibody. Nucleus was counter stained with Dapi. We next determined if the disruption of the lipid raft would block EGF-induced Akt phosphorylation. It has been demonstrated that methyl-β-cyclodextrin (MβCD) can disrupt the cell membrane lipid raft through depletion of cell membrane cholesterol [13]. We treated the cells with different concentrations of MβCD to disrupt the lipid raft, and examined the effects on AKT phosphorylation. We first examined if the lipid raft is disrupted by MβCD, by examining the binding of CT-B to the membrane ( Figure 9A). As shown in Figure 9A, with the increase in MβCD concentration, the binding of CTB to the membrane decreased. CTB was hardly visible when MβCD concentration was at 20 µM. The intensity data indicated in the line scan showed that CT-B intensity (green) dropped from approximately 2000 (no MβCD) to 700 (20 mM MβCD), indicating that the lipid raft is significantly disrupted by 20 µM MβCD ( Figure 9A). The intensity of HER2 was barely changed under all treatment conditions ( Figure 9A). We then examined the effects on EGF-induced Akt phosphorylation. We sho MβCD suppresses AKT phosphorylation in BT474 cells in a dose-dependent man ure 9B), which is consistent with the dose-dependent disruption of lipid rafts ( Fig  Moreover, the inhibitory effects of MβCD on AKT phosphorylation are comparab inhibitory effects of trastuzumab, and the combination of MβCD and trastuzum duced stronger inhibition ( Figure 9C). We then examined the effects on EGF-induced Akt phosphorylation. We showed that MβCD suppresses AKT phosphorylation in BT474 cells in a dose-dependent manner ( Figure 9B), which is consistent with the dose-dependent disruption of lipid rafts ( Figure 9A). Moreover, the inhibitory effects of MβCD on AKT phosphorylation are comparable to the inhibitory effects of trastuzumab, and the combination of MβCD and trastuzumab produced stronger inhibition ( Figure 9C).
Together, our data suggest that Trastuzumab likely inhibits HER2-mediated cell signaling by blocking its translocation to the lipid raft, which is critical for the activation of downstream Akt signaling.

Discussion
To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. While many studies have been performed to reveal how trastuzumab affects the intracellular signaling of breast cancer cells, the data are controversial and fail to provide a clear picture. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. A significant portion of HER2-positive breast cancer cells co-express EGFR and HER3 [91], and HER2-mediated cell signaling, and cell function is closely related to and impacted by other HER receptors [1]. It is not clear how the formation of various HER receptors' homo-and heterodimers impact the function of trastuzumab in HER2-mediated cell signaling, and how this is related to the efficacy of trastuzumab in treating breast cancer. So far, the studies have been focused on the role of HER2-HER3 heterodimers, as HER3 has multiple PI3K binding sites [9,10,92]. However, it is well established that EGFR does not only activate the Ras/Erk pathway, but also strongly activates the PI3K/Akt pathway through multiple mechanisms [8,93,94]. Therefore, we focused our study on the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells.
EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers.
We showed that trastuzumab inhibits EGF-and FBS-induced cell proliferation in both BT474 and SKBR3 cells (Figure 1). This observation is consistent with previous publications [79,95]. Moreover, we showed that trastuzumab blocks cell cycle progression by arresting cells in G1 phase ( Figure 2). Although several studies have shown that trastuzumab arrests the cell cycle at G1 phase [67,69,81,96], no study has assessed the effect of trastuzumab on cell cycle progression in the presence of EGF.
We further showed that trastuzumab does not block EGF-induced EGFR-HER2 heterodimerization ( Figure 3). Differently from pertuzumab, which binds to the HER2 dimerization loop and inhibits HER2 dimerization, trastuzumab binds to the HER2 domain IV, and should not interfere with HER2 dimerization. Indeed, we previously showed that trastuzumab does not block HER2 homodimerization [51]. Other early studies also showed that trastuzumab does not block the heterodimerization of HER2 with EGFR and HER3 [79,95]. Our finding further indicates that trastuzumab does not interfere with HER2 dimerization.
We next studied the effects of trastuzumab on EGF-induced phosphorylation of EGFR and HER2. We showed that trastuzumab does not block EGF-induced phosphorylation of EGFR and HER2 in SKBR3 cells, and actually increases the phosphorylation of EGFR and HER2 in BT474 cells (Figures 4 and 5). When individual tyrosine residues were examined by immunofluorescence, we also showed that trastuzumab does not block the phosphorylation of any of the known phosphotyrosine residues in the HER2 C-terminus including pY1005, pY1112, pY1127, pY1139, pY1196, and pY1248 ( Figure 6). This is not surprising, as we have shown that trastuzumab does not interfere with EGF-induced heterodimerization of EGFR and HER2. Our data are also consistent with previous publications [40][41][42]58,64].
We also studied the effects of trastuzumab on the activation (phosphorylation) of signaling proteins downstream, EGFR and HER2. Previous studies regarding trastuzumab action and resistance in breast cancer have been focused on its role in the Akt pathway, in the context of HRG-induced HER2-HER3 heterodimers [8,60,[67][68][69][70][71][72][73][74][75][76][77]. Very little is known about the effects of trastuzumab on the signaling pathway mediated by EGF-induced EGFR-HER2 heterodimers. We showed that trastuzumab strongly inhibits EGF-induced phosphorylation of Akt at both T308 and S473, and slightly inhibits EGF-induced phosphorylation of Erk (Figure 7). This finding is consistent with our observation that trastuzumab inhibits cell proliferation and cell cycle progression in BT474 and SKBR3 cells (Figures 1 and 2).
However. this finding is quite surprising giving our data regarding the effects of trastuzumab on EGF-induced EGFR and HER2 heterodimerization and phosphorylation (Figures 3-6). How is it that trastuzumab, which specifically binds to HER2, did not block HER2-EGFR dimerization, did not inhibit EGFR and HER2 phosphorylation, yet inhibited Akt phosphorylation and the growth of BT474 and SKBR3 in response to EGF? Indeed, this hypothesis is supported by our data (Figures 8 and 9). We first showed that EGF stimulates the translocation of HER2 and EGFR to the lipid raft, which suggests that lipid raft localization of EGFR/HER2 is involved in their signaling (Figure 8). We further showed that treatment with trastuzumab blocked this EGF-induced translocation of both EGFR and HER2 (Figure 8). This suggests that trastuzumab exerts its effects through EGFR-HER2 heterodimer because HER2 only responds to EGF when it forms a heterodimer with EGFR, and EGFR only responds to trastuzumab when it forms a heterodimer with HER2. These data also indicate that trastuzumab can interfere with EGF-induced cell signaling by blocking the lipid raft translocation of EGFR-HER2 heterodimers.
We finally showed that disrupting the lipid raft with MβCD blocks the localization of HER2 to the lipid raft and blocks Akt phosphorylation in response to EGF (Figure 9). MβCD-induced disruption of the lipid raft and suppression of AKT phosphorylation are parallel in a dose-dependent manner ( Figure 9A,B). Moreover, the effects of MβCD on AKT phosphorylation are comparable to the effects of trastuzumab, and a combination of MβCD and trastuzumab produced stronger inhibition ( Figure 9C). Together, we showed that trastuzumab functions through a novel mechanism: blocking the translocation of EGFR-HER2 heterodimers to the lipid raft that is required for the activation of Akt by the EGFR-HER2 heterodimer.

Conclusions
Trastuzumab does not inhibit EGF-induced dimerization of EGFR and HER2 and does not inhibit EGF-induced phosphorylation of EGFR and HER2, in both BT474 and SKBR3 cells. However, trastuzumab inhibits EGF-induced Akt phosphorylation and cell proliferation by blocking EGF-induced translocation of EGFR-HER2 heterodimers to lipid rafts.