Lapatinib is a human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor (TKI) that has clinical activity in HER2-amplified breast cancer. In vitro studies have shown that lapatinib enhances the effects of the monoclonal antibody trastuzumab suggesting partially non-overlapping mechanisms of action. To dissect these mechanisms, we have studied the effects of lapatinib and trastuzumab on receptor expression and receptor signaling and have identified a new potential mechanism for the enhanced antitumor activity of the combination. Lapatinib, given alone or in combination with trastuzumab to HER2-overexpressing breast cancer cells SKBR3 and MCF7-HER2, inhibited HER2 phosphorylation, prevented receptor ubiquitination and resulted in a marked accumulation of inactive receptors at the cell surface. By contrast, trastuzumab alone caused enhanced HER2 phosphorylation, ubiquitination and degradation of the receptor. By immunoprecipitation and computational protein modeling techniques we have shown that the lapatinib-induced HER2 accumulation at the cell surface also results in the stabilization of inactive HER2 homo- (HER2/HER2) and hetero- (HER2/EGFR and HER2/HER3) dimers. Lapatinib-induced accumulation of HER2 and trastuzumab-mediated downregulation of HER2 was also observed in vivo, where the combination of the two agents triggered complete tumor remissions in all cases after 10 days of treatment. Accumulation of HER2 at the cell surface by lapatinib enhanced immune-mediated trastuzumab-dependent cytotoxicity. We propose that this is a novel mechanism of action of the combination that may be clinically relevant and exploitable in the therapy of patients with HER2-positive tumors.
The human epidermal growth factor receptor 2 (HER2) belongs to the HER family of tyrosine kinase receptors, which also includes HER1 (epidermal growth factor receptor, EGFR), HER3 and HER4. Ligand binding and/or receptor overexpression induces homo- or heterodimerization of HER receptors, transphosphorylation of the kinase domains and subsequent activation of downstream signaling (Tzahar et al., 1996; Yarden and Sliwkowski, 2001; Citri and Yarden, 2006; Sergina et al., 2007). Overexpression/amplification of HER2 is seen in approximately 25–30% of human breast cancers and is associated with a more malignant phenotype and a worse prognosis (Slamon et al., 1987, 1989). Trastuzumab, a humanized monoclonal antibody directed at the extracellular domain of HER2, is active in patients with HER2-overexpressing metastatic breast cancer, reducing relapse-free survival and improving overall survival (Baselga et al., 1996; Slamon et al., 2001; Marty et al., 2005). Recently, trastuzumab has also been found to be efficacious in the adjuvant setting (Piccart-Gebhart et al., 2005; Romond et al., 2005; Slamon et al., 2005). The mechanisms of action of trastuzumab are complex and not fully understood. Described mechanisms include receptor downregulation (Sliwkowski et al., 1999; Ozcelik et al., 2002; Diermeier et al., 2005; Yuste et al., 2005), cell cycle arrest (Kim et al., 2003), inhibition of angiogenesis (Izumi et al., 2002) and induction of antibody-dependent cell-mediated cytotoxicity (ADCC) (Clynes et al., 2000).
Lapatinib, a dual tyrosine kinase inhibitor (TKI) that targets both EGFR and HER2 (Wood et al., 2004; Baselga, 2006), inhibits the growth of HER2-overexpressing breast cancer cells in culture and in tumor xenografts (Chu et al., 2005; Konecny et al., 2006). In the clinic, lapatinib is active and improves time to disease progression in patients with advanced disease who have progressed to trastuzumab (Geyer et al., 2006).
Although trastuzumab and lapatinib provide considerable clinical benefit, a large fraction of HER2-positive tumors display primary resistance to these agents. Even initially sensitive tumors will invariably develop acquired resistance in patients with advanced disease. Therefore, there is a need to develop new strategies to decrease primary resistance and to delay the appearance of acquired secondary resistance. One such approach would be to give the two agents in combination. In preclinical models, the combination is superior to single drug treatment and enhanced apoptosis has been proposed as a mechanism (Xia et al., 2005; Konecny et al., 2006). In the clinic, a phase III study comparing the efficacy of lapatinib versus the combination of lapatinib and trastuzumab in patients with advanced trastuzumab-resistant HER2-positive breast cancer has shown improved clinical outcome with the combination (O’Shaughnessy et al., 2008). In addition, the combined administration of lapatinib and trastuzumab is being studied in a large adjuvant study.
Taking into consideration the promising activity of the combined treatment with trastuzumab and lapatinib, we aimed at exploring further the potential differences between the mechanisms of action of lapatinib and trastuzumab and to search for potential explanations for the enhanced activity of the combination.
Lapatinib induces accumulation of HER2 receptors at the cell surface
Lapatinib treatment of the HER2-overexpressing breast cancer cells SKBR-3 and MCF-7HER2 resulted, as expected, in inhibition of HER2 phosphorylation (data not shown) and inhibition of mitogen-activated protein kinase (MAPK) phosphorylation, a readout of lapatinib inhibition of HER2 signaling ((Scaltriti et al., 2007) and Figure 1a). In terms of total levels of HER2, lapatinib resulted in an accumulation of HER2 when compared with untreated cells (Figure 1a). In a time course experiment we isolated cell surface membrane proteins by whole cell biotin labeling and showed that accumulation of HER2 observed under lapatinib treatment occurred at the plasma membrane, detectable already after 12 h of treatment (Figure 1b).
On the contrary, as reported earlier (Cuello et al., 2001; Valabrega et al., 2005; Henson et al., 2006; Tseng et al., 2006; Scaltriti et al., 2007), trastuzumab alone resulted in overall downregulation of HER2. As for the combined treatment with lapatinib and trastuzumab, the net result was an accumulation of receptor at the cell surface of a similar magnitude to that of lapatinib alone at each time point for MCF-7HER2 cells and starting at 36 h of treatment for SKBR-3 cells. To avoid massive cell death, SKBR-3 cells were treated with lower concentrations of lapatinib and trastuzumab compared with MCF-7HER2 cells. This likely explains the less marked effects in term of HER2 downregulation induced by trastuzumab or HER2 accumulation induced by lapatinib observed in these cells.
Stabilization of HER2 and HER2 dimers in presence of lapatinib
Tyrosine receptor endocytosis and degradation is regulated by post-translational modifications such as receptor phosphorylation and ubiquitination (Marmor and Yarden, 2004). To evaluate the potential role of receptor ubiquitination in lapatinib-induced HER2 accumulation, we transiently expressed hemagglutinin (HA)-tagged ubiquitin in MCF-7HER2 cells and analysed HER2 ubiquitination in presence of lapatinib, trastuzumab or the combination of both. In cells treated with lapatinib alone or in combination with trastuzumab the levels of ubiquitinated receptor were barely detectable (Figure 2a). To determine the turnover rate of HER2 in control cells and in cells treated with either agent alone or the combination, we performed a time course experiment where we metabolically labeled MCF-7HER2 cells with 35S-methionine for 1 h (pulse) and chased the samples at different time points. Cells treated with lapatinib alone or in combination with trastuzumab showed marked HER2 stability (reduced receptor degradation) compared with untreated cells or cells receiving only trastuzumab, with persistence of high levels of HER2 receptor up to 48 h (Figure 2b). On the other hand, consistent with previously reported data (Klapper et al., 2000), trastuzumab treatment markedly increased HER2 ubiquitination (Figure 2a) and degradation (Figure 2b) compared with untreated cells. In addition to the effects on receptor expression, ubiquitination and degradation, we also wanted to study the consequences of lapatinib treatment on the dimerization status of HER2.
In a series of immunoprecipitation experiments, we found that lapatinib enhanced the formation of inactive HER2-containing homodimers and HER2-EGFR and HER2-HER3 heterodimers in both SKBR-3 and MCF-7HER2 cells (Figure 3a and Supplementary Figure 1). The stabilization of HER2-containing dimers was confirmed by cross-linking experiments (Supplementary Figure 1). Quantification of total levels of HER2 and the ratios phospho-tyrosine (pTyr)/HER2 is provided in Figure 3b. Compared with untreated cells, trastuzumab alone resulted in HER2 downregulation and increased p-Tyr/HER2 ratios whereas lapatinib, alone or in combination with trastuzumab, caused accumulation of the receptor with decreased p-Tyr/HER2 ratios. Consistent with the results shown in Figure 1, these effects were more marked in MCF-7HER2 cells. As above, all the experiments were repeated three times.
Modeling of lapatinib-binding affinity to HER receptors
Lapatinib competes with ATP for binding to the kinase domain of both HER2 and EGFR. Given the biochemical data on lapatinib-dependent HER2 dimer stabilization, we opted for a structural modeling approach to measure the energy gain (degree of stabilization) of HER2 dimers associated with lapatinib versus HER2 dimers associated with ATP. We computed and compared the affinities of both lapatinib and ATP for the monomeric and dimeric forms of the kinase domains of the members of EGFR, HER2 and HER3 (HER3 only binds ATP). Although structural data is only available for the kinase domain of EGFR, the close similarity in sequences of the other family members (the sequences of HER2 and HER3 catalytic domains are 77.7 and 56.7% identical respectively, to EGFR) enabled us to construct reliable structural models for the other members based on homology. The manner in which the domains are thought to dimerize, leading to activation, (asymmetric dimerization (Zhang et al., 2006)) is shown in Figure 4a. In agreement with experimental observations (Rusnak et al., 2001), our calculations show that: (a) lapatinib has a higher affinity for HER2 monomers than it does for EGFR monomers (Figure 4b), (b) lapatinib has higher affinity than ATP for HER2 monomers (Figure 4b) (c) the HER2 dimers (specifically HER2 homodimers and heterodimers with EGFR and HER3) are more stable in the presence of lapatinib (Figure 4c).
Effects of lapatinib and trastuzumab on BT474 xenografts
To expand our results in vivo, we evaluated tumor growth inhibition and HER2 expression in xenografts derived from BT474 cells in response to lapatinib, trastuzumab or the combination. BT474 cells were used as they are highly tumorigenic and sensitive to both lapatinib and trastuzumab. Treatments were started on day 13 post injection, when tumors were already established. On day 19, we sacrificed two animals per group for immunohistochemistry analyses and the experiment continued on the remaining animals until day 23.
As expected (Baselga et al., 1998; Konecny et al., 2006), both lapatinib and trastuzumab induced tumor regression of BT474 cell-derived xenografts. All the mice receiving the combination of lapatinib and trastuzumab showed complete tumor remission after 10 days (day 23) of treatment (Figure 5a). In these animals no tumor relapse was observed after 8 months from the completion of therapy. Tumors derived from the animals excluded at day 19 (6 days of treatment) were excised and subjected to anti-HER2 immunohistochemistry analysis. There was an increase in HER2 expression in tumors treated with lapatinib alone and a decrease of HER2 expression in trastuzumab-treated tumors as compared with controls (Figure 5b). The degree of decrease of HER2 expression did not reach its peak at this point as treatment with trastuzumab for 16 days resulted in a higher degree of HER2 downregulation (data not shown). In the combination group, the effects of lapatinib on HER2 accumulation were dominant over those of trastuzumab (Figure 5b). Quantification of the HER2 membrane staining is expressed as a mean of 10 representative fields for each condition (animals were obtained from three independent experiments, Figure 5c).
Lapatinib-induced accumulation of inactive HER2 leads to increased ADCC in vitro
Engagement of immune-effector systems is one of the main therapeutic mechanisms of anti-HER antibodies (Clynes et al., 2000; Bleeker et al., 2004; Gennari et al., 2004). Thus, we wanted to test whether the accumulation of HER2 induced by lapatinib could increase trastuzumab-dependent cell cytotoxicity in MCF-7HER2 cells by increasing the number of antibody binding sites at the cell surface. As shown in Figure 6, trastuzumab-dependent cytotoxicity was significantly higher in MCF-7HER2 cells treated with lapatinib compared with untreated cells. To validate this result in a different model system, we measured trastuzumab-mediated cytotoxicity in cells expressing low levels of HER2 (MCF-7IRES) and in cells where the effect of lapatinib on HER2 accumulation was mimicked by stable expression of medium levels of kinase inactive receptor (MCF-7HER2KD). The cells with higher HER2 expression showed significantly higher trastuzumab-mediated cytolysis (data not shown). The results are expressed as mean of three independent experiments
We have shown that lapatinib, a small molecule HER2 TKI, prevents HER2 ubiquitination and degradation, which in turn results in a substantial accumulation of inactive HER2 receptors at the cytoplasmic membrane. This lapatinib-induced accumulation of HER2 was also observed in vivo. These effects remain even in the presence of trastuzumab that has opposite effects on receptor ubiquitination and degradation when given alone. The degree to which HER2 receptors are internalized and downregulated following treatment with anti-HER2 antibodies is still a matter of debate. Although some groups have reported receptor downregulation (Cuello et al., 2001; Valabrega et al., 2005; Henson et al., 2006; Tseng et al., 2006; Scaltriti et al., 2007), others have not (Austin et al., 2004; Hommelgaard et al., 2004; Longva et al., 2005). Interestingly, in some of the models that have not observed trastuzumab-mediated receptor downregulation (Austin et al., 2004; Longva et al., 2005), there is also a lack of trastuzumab-induced HER2 phosphorylation. It is therefore plausible that kinase activation is a required step for receptor ubiquitination and degradation and that, as a consequence, lapatinib and other receptor TKIs prevent receptor downregulation.
Our computational modeling and immunoprecipitation experiments showed that lapatinib provides stability to HER2 dimers and prolongs the half-live of these inactive, dimerized HER2 receptors. Inactive EGFR/HER dimers also occur after therapy with EGFR TKIs (Anido et al., 2003). The binding of these agents to the ATP pocket of the receptor perturbs its three-dimensional structure, stabilizing interactions among receptors and promoting the accumulation of inactive EGFR dimers (Arteaga et al., 1997; Gan et al., 2007). The presence of high levels of EGFR inactive dimers on the cell surface would also act as a ligand trap, being able to bind (and sequester) the ligands without consequent receptor phosphorylation. Addition of anti-EGFR antibodies would improve the efficacy of TKIs, as it would keep the receptor inactive once the TKIs disassociate. This model provides a possible explanation for the efficacy of the combination of TKIs with anti-EGFR antibodies, especially in conditions when the ligands are present in limiting amounts (Arteaga et al., 1997; Lichtner et al., 2001; Gan et al., 2007). Besides the enhanced receptor stability, it is possible that the increased receptor number could also play a role in the observed enhanced co-immunoprecipitation of EGFR or HER3 with HER2. Taken together, inhibition of phosphorylation and ubiquitination followed by stabilization of inactive HER dimers and the resulting increase in receptor number may be a general modus operandi of small molecule TKIs targeting the HER family.
In our studies we have not analysed the potential mechanism responsible for HER2 ubiquination and degradation. A key regulator of HER receptor degradation is the E3 ubiquitin ligase c-Cbl (Marmor and Yarden, 2004). Although E3 ubiquitin ligase c-Cbl shows only a marginal effect in ligand-induced HER2 ubiquitination (Wang et al., 1999; Hommelgaard et al., 2004), it does play a role in receptor degradation when c-Cbl is overexpressed (Li et al., 2007) or recruited following treatment with anti-HER2 antibodies (Klapper et al., 2000; Wolpoe et al., 2003). However, we cannot rule out that the internalization of HER2 following trastuzumab treatment could be mediated by a kinase-dependent activation of other ubiquitin ligases.
We have identified an alternative potential mechanism for the enhanced effect of combined therapy with a TKI and an anti-ErbB antibody. The accumulation of inactive HER2 receptor at the cell surface may lead to enhanced or prolonged trastuzumab binding/activity, which in turn could explain the observed increase in trastuzumab-mediated ADCC. ADCC is dependent on both antibody affinity and expression levels of the target receptor; target tumor cells with higher antigen expression are more susceptible to antibody therapy due to enhanced immune effects (Mimura et al., 2005; Tang et al., 2007). In our experiments, this was found to be the case as there was a good correlation between HER2 levels and ADCC, both in HER2-overexpressing cells treated with lapatinib and in cells transfected with a kinase dead HER receptor (MCF7-HER2KD). We are now studying the role of ADCC in vivo where the therapy with the combination resulted in a remarkable and rapid complete regression of well-established xenografts in all treated animals.
In the clinic, there is also growing evidence that trastuzumab's antitumor activity may be partially mediated by ADCC. For example, in a pilot presurgical trastuzumab study, patients who achieved either a partial or a complete response to trastuzumab were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro ADCC activity (Gennari et al., 2004). There is also a suggestion that patients with certain polymorphisms of their FcγRIIIA receptors, which are activating antibody receptors present on the effector cells responsible for trastuzumab and other antibody-mediated ADCC, may have an enhanced response to trastuzumab (Musolino et al., 2008). The addition of lapatinib to trastuzumab could be therefore particularly active in patients with given FcγRIIIA genotypes such as FcγRIIIA-158 V/V.
It is also conceivable that prolonged trastuzumab administration results in a decrease in the total levels of surface HER2 in breast tumors in a similar fashion as it occurs in preclinical models. An interesting study showed that in tumors trastuzumab caused a decrease in HER2 expression while maintaining the levels of gene amplification by FISH, indicating that the phenomenon was due to true protein downregulation rather than selective elimination of HER2-positive cells (Milella et al., 2004). In this regard, it has been recently shown that increased receptor ubiquitination and downregulation plays a role in acquired resistance to antibody-based antireceptor therapy (Lu et al., 2007). Lapatinib could delay/counteract this occurrence by increasing HER2 expression levels and, as a consequence, prevent or delay trastuzumab resistance due to lower HER2 expression.
Finally, the concept of combining an antireceptor monoclonal antibody and a receptor-stabilizing TKI could be expanded to other members of the HER receptor family. At least three independent groups, including ours, have shown additional or synergistic antitumor effects using the combination of different anti-EGFR antibodies with TKIs in targeting EGFR-positive cells (Johns et al., 2003; Huang et al., 2004; Matar et al., 2004; Perera et al., 2005). In further support of this approach we have observed promising clinical activity of the combination of an EGFR TKI and cetuximab, a monoclonal antibody directed at the extracellular domain of the EGFR (Baselga et al., 2006).
In conclusion, our results provide a new explanation for the enhanced effects of the combination of lapatinib and trastuzumab. Lapatinib reduces HER2 ubiquination, prevents HER2 degradation, and induces the formation of inactive HER2 dimers at the cell surface, which in turn provides an increase in trastuzumab binding and a greater trastuzumab-mediated immune response (Figure 7). This is a therapeutically exploitable mechanism of action that deserves further study in patients.
Materials and methods
Cell lines and treatments
MCF-7 HER2 (overexpressing HER2), MCF-7HER2KD (KD: Kinase Dead; expressing kinase inactive HER2) and MCF-7IRES (mock transfected) cells were obtained as described earlier (Scaltriti et al., 2007). SKBR-3 (HER2 amplified) and MDA-MB-468 (HER2 negative) cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in Dulbecco's modified Eagle's medium/Ham F12 1:1 (DMEM/F12) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (Life Technologies Inc. Ltd., Paisley, UK) at 37 °C in 5% CO2. MCF-7 HER2, MCF-7HERKD and MCF-7IRES cells were maintained in the same medium containing 30 μg/ml hygromycin B (Life Technologies Inc.).
Trastuzumab (Herceptin; kindly provided by F Hoffmann-La Roche, Basel, Switzerland) and Cetuximab (Erbitux; kindly provided by Merck KGaA, Darmstadt, Germany) were dissolved in sterile apyrogen water and stored at 4 °C. Lapatinib (Tykerb; kindly provided by GlaxoSmithKline, Research Triangle Park, NJ, USA) was dissolved in dimethyl sulfoxide (dimethyl sulfoxide as a stock solution at 10 mM) and stored at −20 °C. MCF-7 HER2 cells were treated with trastuzumab and lapatinib at a final concentration of 100 nM and 1 μM in the culture media, respectively. SKBR-3 cells were treated with trastuzumab and lapatinib at a final concentration of 20 and 100 nM in the culture media, respectively. Dimethyl sulfoxide (equal volume to that of treated cells) was added to culture media of the control cells.
Biotin pull down, protein immunoprecipitation, protein cross-linking and western blot
For biotin pull down assays, cells were grown in 60 mm dishes and treated with either trastuzumab, lapatinib or the combination for the indicated times. Cells were incubated with EZ-LINK Sulfo-Biotin (Pierce, Rockford, IL, USA) for 2 h at 4 °C with gentle rotation. The reaction was stopped by washing twice with 25 nM Tris-Hcl (pH 7.5) in PBS (phosphate-buffered saline) and cells were scraped into ice-cold lysis buffer (50 mmol/l HEPES, pH 7.0, 10% glycerol, 1% Triton X-100, 5 mmol/l EDTA (ethylenediaminetetraacetic acid), 1 mmol/l MgCl2, 25 mmol/l NaF, 50 μg/ml leupeptin, 50 μg/ml aprotinin, 0.5 mmol/l orthovanadate, and 1 mmol/l phenylmethylsulfonyl fluoride). Lysates were centrifuged at 15 000 g for 20 min at 4 °C, and supernatants were removed and assayed for protein concentration using the Dc Protein assay (Bio-Rad, CA, USA). A volume of 500 μl of lysis buffer containing equal amount of proteins was incubated with UltraLink Immobilized NeutrAvidin protein (Pierce Rockford, IL, USA) 2 h at 4 °C with gentle rotation and washed three times with lysis buffer before suspension in SDS (sodium dodecyl sulfate)-loading buffer.
For immunoprecipitation experiments, cells were grown in 100 mm dishes and treated with either trastuzumab, lapatinib or the combination for 48 h. A volume of 500 μl of lysis buffer containing equal amount of proteins was incubated with 10 μg trastuzumab for HER2 precipitation overnight at 4 °C with gentle rotation. Protein A sepharose beads (Amersham Biosciences, Uppsala, Sweden) were added for 2 h and washed three times with lysis buffer before suspension in SDS-loading buffer. For cross-linking experiments, cells were grown in 100 mm dishes and treated with trastuzumab, lapatinib or the combination for 48 h. Cells were detached using 10 mM EDTA in PBS and gentle scraping, and incubated in 5 mM bis(sulfosuccinimidyl) suberate (BS3) for 30 min at room temperature with gentle rotation. Cross-linking reaction was stopped by incubating cells in 25 mM Tris-HCl for 15 min at room temperature with gentle rotation. Cells were then processed for immunoprecipitation with 10 μg trastuzumab as described above.
For immunoblots, total lysates, biotin pull down and immunoprecipitation extracts were resolved by SDS–PAGE (polyacrylamide gel electrophoresis) on either 8% (for phosphotyrosine HER2 and HER3 detection) or 12% (for phospho-MAPKs (p-MAPKs) and total MAPKs detection) acrylamide, and electrophoretically transferred to nitrocellulose membranes. For cross-linking experiments, precast gradient 4–15% Tris-HCl gels (READY GEL Bio-Rad, CA, USA) were used. Membranes were hybridized with the following primary antibodies: mouse monoclonal anti-p-Tyr (clone 4G10, cat: 05-321) and mouse monoclonal anti-total HER3 (clone 2F12, cat: 05-390; Upstate Lake Placid, NY, USA), rabbit polyclonal anti-total EGFR (Abcam, Cambridge, UK), mouse monoclonal anti-total HER2 (CB11, Biogenex, San Ramon, CA, USA), mouse monoclonal anti-transferrin receptor (Zymed Laboratories, San Francisco, CA, USA), rabbit polyclonal phospho-p44/42 MAPK (Thr202/Tyr204) and rabbit polyclonal total MAPKs (Cell Signaling Technology, Beverly, MA, USA). Anti-p-Tyr, anti-EGFR, anti-HER2 and anti-HER3 antibodies were incubated in Tris-buffered saline-Tween buffer (T-TBS, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20)/5% non-fat dry milk. Anti-p-MAPKs and anti-total MAPKs were incubated in T-TBS/5% bovine serum albumin. Protein–antibody complexes were detected by chemiluminescence with the SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA), and images were captured with a FUJIFILM LAS-3000 camera system. Densitometric analyses for protein quantification were done using Image J 1.38x software (http://rsbweb.nih.gov/ij/index.html). The experiments were repeated at least three times.
MCF-7HER2 cells were transfected with HA-ubiquitin vector (gift from Dr José Gonzales Castaño) using the non-liposomal FuGENE 6 reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol. Briefly, 60 mm dishes (at 50% density) were transfected with 4 μg of plasmid and treated, after 24 h, with trastuzumab, lapatinib or the combination for 6 h in the presence of 10 μM MG-132 proteasome and calpain inhibitor (Sigma, St Louis, MI, USA). A volume of 500 μl of lysis buffer containing equal amount of proteins was incubated with trastuzumab for HER2 immunoprecipitation. Samples were resolved and electrophoretically transferred to nitrocellulose membranes as described above and blotted with anti-HA antibody (anti-HA hybridome, 1:100, Babco, Richmond, CA, USA) overnight at 4 °C.
Metabolic labeling (pulse chase)
Dishes of MCF-7HER2 cells (60 mm) were preincubated 4 h in serum-free Dulbecco's modified Eagle's medium deprived of Met and Cys and metabolically labeled for 1 h with the same medium containing 20 μCi/dish of 35S-Translabel (MP Biomedicals, Irvine, CA, USA). Treatments with trastuzumab, lapatinib or the combination were carried out in 10% serum containing DMEM-F12 medium. After lysis and HER2 immunoprecipitation with trastuzumab, samples were analysed by SDS–PAGE and autoradiography.
Tumor xenografts in nude mice
Mice (Charles Rivers Laboratories, Paris, France) were maintained and treated as described earlier (Scaltriti et al., 2007). A 17β-estradiol pellet (Innovative Research of America, Sarasota, FL, USA) was inserted subcutaneously to each mouse 1 day before cell injection. BT474 VH2 cells were obtained from in vitro explants of BT474-derived xenografts (Baselga et al., 1998). A total of 2 × 107 cells were injected into the right flanks of 48 mice (12 for each experimental condition), and treatment began when tumors reached an average size of >600 mm3 (13 days after injection). Trastuzumab (10 mg/kg in sterile PBS) or sterile PBS (control) was given intraperitoneally twice weekly. Lapatinib (100 mg/kg) was administered daily by oral gavage in 0.5% hydroxypropyl methylcellulose, 0.1% Tween 80. Tumor xenografts were measured with calipers three times a week, and tumor volume was determined using the formula: (length × width2) × (π/6). After 10 days of treatment the animals were anesthetized with 1.5% isoflurane–air mixture and killed by cervical dislocation. Results are presented as mean±s.d. The experiments were repeated three times.
Xenografts samples were prepared as described earlier (Serra et al., 2008). Primary antibody was anti-HER2 (CB11, Biogenex) and secondary antibody was from Amersham. As a negative control, primary antibody was omitted. Slides were scanned with ScanScope CS system (Aperio, Vista, CA, USA) and HER2 staining intensity was quantified by PATHIAMRUO software (BioImagene Inc, San Mateo, CA, USA).
Antibody-dependent cell-mediated cytotoxicity assay
Antibody-dependent cell-mediated cytotoxicity was measured with the CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, WI, USA) according to manufacturer's instructions. Briefly, MCF-7HER2, MCF-7IRES and MCF-7HER2KD cells were used as target cells. Peripheral blood mononuclear cells obtained from a healthy donor were used as effector cells. In all, 4 × 103 MCF-7HER2 cells were seeded in triplicate for each condition in a 96-well plate, treated 48 h with 1 μM lapatinib and, in the presence or absence of 8 × 103 viable peripheral blood mononuclear cells, incubated with trastuzumab (100 nM) for 4 h. MCF-7IRES and MCF-7HER2KD cells were not previously treated with lapatinib. Viability of peripheral blood mononuclear cells was assessed by Guava PCA using Guava ViaCount reagents (Guava Technologies, Hayward, CA, USA). The percentage of cytotoxicity was calculated after correcting for background absorbance values according to the following formula:
Specificity of trastuzumab in causing immune-mediated cytolysis was ensured performing the same assays with cetuximab, an anti-EGFR therapeutic antibody. In all the conditions, cetuximab-dependent cytotoxicity was lower than 5%. MDA-MB-468 cells (HER2 negative) served as negative control for trastuzumab ADCC. Results are presented as means±s.d. Each experiment was repeated three times.
Computational protein modeling
The EGFR-ATP complex model was constructed guided by the structure of cAMP-dependent kinase (PDB code 1ATP (Zheng et al., 1993)) and of EGFR (PDB code 1M14 (Zhang et al., 2006)) using QUANTA (Accelrys, San Diego, CA, USA). The activated EGFR asymmetric and symmetric dimers (Zhang et al., 2006) were generated from the monomer using crystallographic symmetry operators. The HER2 and HER3 sequences were aligned with that of EGFR and, using the structure of the monomeric EGFR-ATP complex as a template, the structures of HER2 and HER3 were built using the MODELLER program (Sali and Blundell, 1993). Homo- and heterodimeric models of EGFR, HER2 and HER3 in their active and inactive states were generated by superposition of the modeled monomers against the EGFR dimer. Complexes with lapatinib were constructed based on the EGFR–lapatinib complex (PDB code 1XKK (Wood et al., 2004)). All models were optimized using CHARMM (Brooks et al., 1983) and minimized until the gradient of potential energy was smaller than 10−2 kcal/mol/Å.
For in vitro assays and nude mice experiments, comparisons between groups were made using a two-tailed Student’s t-test. Differences for which P was less than 0.05 were considered statistically significant.
Anido J, Matar P, Albanell J, Guzman M, Rojo F, Arribas J et al. (2003). ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells. Clin Cancer Res 9: 1274–1283.
Arteaga CL, Ramsey TT, Shawver LK, Guyer CA . (1997). Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site. J Biol Chem 272: 23247–23254.
Austin CD, De Maziere AM, Pisacane PI, van Dijk SM, Eigenbrot C, Sliwkowski MX et al. (2004). Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 15: 5268–5282.
Baselga J . (2006). Targeting tyrosine kinases in cancer: the second wave. Science 312: 1175–1178.
Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J . (1998). Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 58: 2825–2831.
Baselga J, Schoffski F, Rojo F, Dumez H, Ramos FL, Macarulla T et al. (2006). A phase I pharmacokinetic (PK) and molecular pharmacodynamic (PD) study of the combination of two anti-EGFR therapies, the monoclonal antibody (MAb) cetuximab (C) and the tyrosine kinase inhibitor (TKI) gefitinib (G), in patients (pts) with advanced colorectal (CRC), head and neck (HNC) and non-small cell lung cancer (NSCLC). Journal of Clinical Oncology, 2006 ASCO Annual Meeting Proceedings Part I. Vol 24, No. 18S (June 20 Supplement), 2006: 3006.
Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L et al. (1996). Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J Clin Oncol 14: 737–744.
Bleeker WK, Lammerts van Bueren JJ, van Ojik HH, Gerritsen AF, Pluyter M, Houtkamp M et al. (2004). Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy. J Immunol 173: 4699–4707.
Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M . (1983). CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comp Chem 4: 187–217.
Citri A, Yarden Y . (2006). EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7: 505–516.
Clynes RA, Towers TL, Presta LG, Ravetch JV . (2000). Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 6: 443–446.
Cuello M, Ettenberg SA, Clark AS, Keane MM, Posner RH, Nau MM et al. (2001). Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res 61: 4892–4900.
Chu I, Blackwell K, Chen S, Slingerland J . (2005). The dual ErbB1/ErbB2 inhibitor, lapatinib (GW572016), cooperates with tamoxifen to inhibit both cell proliferation- and estrogen-dependent gene expression in antiestrogen-resistant breast cancer. Cancer Res 65: 18–25.
Diermeier S, Horvath G, Knuechel-Clarke R, Hofstaedter F, Szollosi J, Brockhoff G . (2005). Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation. Exp Cell Res 304: 604–619.
Gan HK, Walker F, Burgess AW, Rigopoulos A, Scott AM, Johns TG . (2007). The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 increases the formation of inactive untethered EGFR dimers. Implications for combination therapy with monoclonal antibody 806. J Biol Chem 282: 2840–2850.
Gennari R, Menard S, Fagnoni F, Ponchio L, Scelsi M, Tagliabue E et al. (2004). Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res 10: 5650–5655.
Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T et al. (2006). Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 355: 2733–2743.
Henson ES, Hu X, Gibson SB . (2006). Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression. Clin Cancer Res 12: 845–853.
Hommelgaard AM, Lerdrup M, van Deurs B . (2004). Association with membrane protrusions makes ErbB2 an internalization-resistant receptor. Mol Biol Cell 15: 1557–1567.
Huang S, Armstrong EA, Benavente S, Chinnaiyan P, Harari PM . (2004). Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor. Cancer Res 64: 5355–5362.
Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK . (2002). Tumour biology: herceptin acts as an anti-angiogenic cocktail. Nature 416: 279–280.
Johns TG, Luwor RB, Murone C, Walker F, Weinstock J, Vitali AA et al. (2003). Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478. Proc Natl Acad Sci USA 100: 15871–15876.
Kim JY, Sun Q, Oglesbee M, Yoon SO . (2003). The role of ErbB2 signaling in the onset of terminal differentiation of oligodendrocytes in vivo. J Neurosci 23: 5561–5571.
Klapper LN, Waterman H, Sela M, Yarden Y . (2000). Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res 60: 3384–3388.
Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M et al. (2006). Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res 66: 1630–1639.
Li X, Shen L, Zhang J, Su J, Liu X, Han H et al. (2007). Degradation of HER2 by Cbl-based chimeric ubiquitin ligases. Cancer Res 67: 8716–8724.
Lichtner RB, Menrad A, Sommer A, Klar U, Schneider MR . (2001). Signaling-inactive epidermal growth factor receptor/ligand complexes in intact carcinoma cells by quinazoline tyrosine kinase inhibitors. Cancer Res 61: 5790–5795.
Longva KE, Pedersen NM, Haslekas C, Stang E, Madshus IH . (2005). Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down-regulation of ErbB2. Int J Cancer 116: 359–367.
Lu Y, Li X, Liang K, Luwor R, Siddik ZH, Mills GB et al. (2007). Epidermal growth factor receptor (EGFR) ubiquitination as a mechanism of acquired resistance escaping treatment by the anti-EGFR monoclonal antibody cetuximab. Cancer Res 67: 8240–8247.
Marmor MD, Yarden Y . (2004). Role of protein ubiquitylation in regulating endocytosis of receptor tyrosine kinases. Oncogene 23: 2057–2070.
Marty M, Cognetti F, Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin M et al. (2005). Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group. J Clin Oncol 23: 4265–4274.
Matar P, Rojo F, Cassia R, Moreno-Bueno G, Di Cosimo S, Tabernero J et al. (2004). Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting. Clin Cancer Res 10: 6487–6501.
Milella M, Trisciuoglio D, Bruno T, Ciuffreda L, Mottolese M, Cianciulli A et al. (2004). Trastuzumab down-regulates Bcl-2 expression and potentiates apoptosis induction by Bcl-2/Bcl-XL bispecific antisense oligonucleotides in HER-2 gene—amplified breast cancer cells. Clin Cancer Res 10: 7747–7756.
Mimura K, Kono K, Hanawa M, Kanzaki M, Nakao A, Ooi A et al. (2005). Trastuzumab-mediated antibody-dependent cellular cytotoxicity against esophageal squamous cell carcinoma. Clin Cancer Res 11: 4898–4904.
Musolino A, Naldi N, Bortesi B, Pezzuolo D, Capelletti M, Missale G et al. (2008). Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J Clin Oncol 26: 1789–1796.
O’Shaughnessy J, Blackwell KL, Burstein H, Storniolo AM, Sledge G, Baselga J et al. (2008). A randomized study of lapatinib alone or in combination with trastuzumab in heavily pretreated HER2+ metastatic breast cancer progressing on trastuzumab therapy. J Clin Oncol 26 (Suppl): abstract 1015.
Ozcelik C, Erdmann B, Pilz B, Wettschureck N, Britsch S, Hubner N et al. (2002). Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc Natl Acad Sci USA 99: 8880–8885.
Perera RM, Narita Y, Furnari FB, Gan HK, Murone C, Ahlkvist M et al. (2005). Treatment of human tumor xenografts with monoclonal antibody 806 in combination with a prototypical epidermal growth factor receptor-specific antibody generates enhanced antitumor activity. Clin Cancer Res 11: 6390–6399.
Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I et al. (2005). Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353: 1659–1672.
Romond EH, Perez EA, Bryant J, Suman VJ, Geyer Jr CE, Davidson NE et al. (2005). Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353: 1673–1684.
Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N et al. (2001). The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 1: 85–94.
Sali A, Blundell TL . (1993). Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234: 779–815.
Scaltriti M, Rojo F, Ocana A, Anido J, Guzman M, Cortes J et al. (2007). Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst 99: 628–638.
Sergina NV, Rausch M, Wang D, Blair J, Hann B, Shokat KM et al. (2007). Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 445: 437–441.
Serra V, Markman B, Scaltriti M, Eichhorn PJ, Valero V, Guzman M et al. (2008). NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res 68: 8022–8030.
Slamon D, Eiermann W, Robert N, Pienkowski T, Martin M, Pawlicki M et al. (2005). Phase III randomized trial comparing doxorubicin and cyclophosphamide followed by docetaxel (ACT) with doxorubicin and cyclophosphamide followed by docetaxel and trastuzumab (ACTH) with docetaxel, carboplatin and trastuzumab (TCH) in HER2 positive early breast cancer patients: BCIRG 006 study. 28th Annual San Antonio Breast Cancer Symposium abstract 1. San Antonio, TX, USA (abstract 1).
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL . (1987). Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177–182.
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE et al. (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707–712.
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A et al. (2001). Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344: 783–792.
Sliwkowski MX, Lofgren JA, Lewis GD, Hotaling TE, Fendly BM, Fox JA . (1999). Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol 26: 60–70.
Tang Y, Lou J, Alpaugh RK, Robinson MK, Marks JD, Weiner LM . (2007). Regulation of antibody-dependent cellular cytotoxicity by IgG intrinsic and apparent affinity for target antigen. J Immunol 179: 2815–2823.
Tseng PH, Wang YC, Weng SC, Weng JR, Chen CS, Brueggemeier RW et al. (2006). Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor. Mol Pharmacol 70: 1534–1541.
Tzahar E, Waterman H, Chen X, Levkowitz G, Karunagaran D, Lavi S et al. (1996). A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 16: 5276–5287.
Valabrega G, Montemurro F, Sarotto I, Petrelli A, Rubini P, Tacchetti C et al. (2005). TGFalpha expression impairs Trastuzumab-induced HER2 downregulation. Oncogene 24: 3002–3010.
Wang Z, Zhang L, Yeung TK, Chen X . (1999). Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation. Mol Biol Cell 10: 1621–1636.
Wolpoe ME, Lutz ER, Ercolini AM, Murata S, Ivie SE, Garrett ES et al. (2003). HER-2/neu-specific monoclonal antibodies collaborate with HER-2/neu-targeted granulocyte macrophage colony-stimulating factor secreting whole cell vaccination to augment CD8+ T cell effector function and tumor-free survival in Her-2/neu-transgenic mice. J Immunol 171: 2161–2169.
Wood ER, Truesdale AT, McDonald OB, Yuan D, Hassell A, Dickerson SH et al. (2004). A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res 64: 6652–6659.
Xia W, Gerard CM, Liu L, Baudson NM, Ory TL, Spector NL . (2005). Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells. Oncogene 24: 6213–6221.
Yarden Y, Sliwkowski MX . (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127–137.
Yuste L, Montero JC, Esparis-Ogando A, Pandiella A . (2005). Activation of ErbB2 by overexpression or by transmembrane neuregulin results in differential signaling and sensitivity to herceptin. Cancer Res 65: 6801–6810.
Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J . (2006). An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125: 1137–1149.
Zheng JH, Trafny EA, Knighton DR, Xuong NH, Taylor SS, Ten Eyck LF et al. (1993). 2.2-angstrom refined crystal-structure of the catalytic subunit of cAMP-dependent protein-kinase complexed with MnATP and a peptide inhibitor. Acta Crys D 49: 362–365.
This work was supported in full by a grant of the Breast Cancer Research Foundation.
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Scaltriti, M., Verma, C., Guzman, M. et al. Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity. Oncogene 28, 803–814 (2009). https://doi.org/10.1038/onc.2008.432
- ErbB receptor
- tyrosine kinase inhibitor
- antibody-dependent cell cytotoxicity (ADCC)
- breast cancer
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