Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity

Abstract

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.

Introduction

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.

Results

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).

Figure 1
figure1

Lapatinib induces HER2 accumulation. (a) Western blot showing HER2, phospho-MAPKs (p-MAPKs) and total MAPKs (loading control) expression in total lysates of both SKBR-3 and MCF-7HER2 cells treated 48 h with trastuzumab (T), lapatinib (L) or the combination (T+L). Untreated cells served as controls (C). Western blot bands were quantified by Image J (NIH) and HER2 levels of treated cells (normalized to MAPKs) from three experiments were expressed as arbitrary units±s.e.m. relative to controls. (b) Western blot to detect membrane-localized HER2 in both SKBR-3 and MCF-7HER2 cells treated for 12, 24, 36 and 48 h with trastuzumab, lapatinib or the combination. Whole cells were labeled with biotin and membrane bound proteins were pulled down with NeutrAvidin beads. IB, immunoblot. HER2 levels of treated cells (normalized to transferring receptor (TfR)) were quantified and expressed as described above.

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.

Figure 2
figure2

Effects of lapatinib on HER2 ubiquitination and stabilization. (a) Western blot showing both ubiquitinated (HA) and total HER2 in MCF-7HER2 cells previously transfected with HA-ubiquitin and treated with trastuzumab (T), lapatinib (L) or the combination (T+L) for 6 h in presence of 10 μM MG-132 proteasome and calpain inhibitor. Untreated cells served as controls (C). HA levels of treated cells (normalized to total HER2) were quantified and expressed as arbitrary units±s.e.m. relative to controls. IP, immunoprecipitation; IB, immunoblot. (b) Autoradiography detecting 35S-labeled immunoprecipitated HER2 in MCF-7HER2 cells metabolically pulsed for 1 h and chased after 12, 24, 36 and 48 h of treatment with trastuzumab (T), lapatinib (L) or the combination (T+L). The first lane indicates the amount of labeled HER2 after 1 h of pulse. 35S-labeled HER2 levels of treated cells were quantified and expressed as arbitrary units±s.e.m. relative to controls. The experiments were repeated three times.

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.

Figure 3
figure3

Lapatinib promotes HER2 dimerization. (a) Western blot detecting phospho-tyrosine (p-Tyr), EGFR, HER2 and HER3 in both SKBR-3 and MCF-7HER2 cells treated with trastuzumab (T), lapatinib (L) or the combination (T+L) for 48 h and subjected to immunoprecipitation (IP) with an anti-HER2 antibody. Untreated cells served as controls (C). Total lysates were probed for p-MAPKs and total MAPKs (loading control). (b) Western blot quantification of HER2 (normalized to MAPKs) and the ratios (p-Tyr)/HER2 of treated cells expressed as arbitrary units±s.e.m. relative to controls. 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).

Figure 4
figure4

Effect of lapatinib binding on HER2 dimer stabilization. (a) Example of HER2 dimerization modeling. Asymmetric mode of dimerization of the HER family kinase domains structurally modeled in this study. The dimer between HER2 (in red and complexed with lapatinib) and HER3 (in magenta and complexed with ATP and associated magnesium and water molecules) is shown as an example. (b) Computed energy differences between the binding of lapatinib and of ATP respectively to HER monomers. Values reflect the degree of stabilization of HER monomers bound to lapatinib (left column) versus HER monomers bound to ATP (right column). LAP, lapatinib; ATP, Adenosine 5′-triphosphate; i, inactive conformation; a, active conformation. (c) Computed energy differences between the binding of lapatinib and of ATP respectively to HER2 dimers. Values reflect the degree of stabilization of HER2 dimers bound to lapatinib (left column) versus HER2 dimers bound to ATP (right column). *Lapatinib does not bind HER3. LAP, lapatinib; ATP, Adenosine 5′-triphosphate; i, inactive conformation; a, active conformation.

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).

Figure 5
figure5

Antitumor activity of lapatinib and trastuzumab on BT474 xenografts. (a) Tumor growth inhibition in response to trastuzumab, lapatinib or the combination of the two agents. Treatments started at day 13. Student's t-test was used to compare tumor sizes between the groups and data are expressed as mean±s.d. *P<0.05, **P<0.01 versus control; #P<0.05 versus trastuzumab; ##P<0.01 versus both lapatinib and trastuzumab. These differences remained statistically significant for the entire duration of the experiment. The experiment was performed three times with similar results. (b) Representative immunohistochemistry showing HER2 expression in tumors xenografts treated as indicated and sacrificed at day 19 (see panel a). (c) Quantification of the median intensity of the completely stained cells expressed as mean of 10 representative fields for each condition. Student's t-test: *P<0.05, **P<0.01 versus control; #P<0.05 versus trastuzumab.

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

Figure 6
figure6

Trastuzumab-dependent cell-mediated cytotoxicity. ADCC mediated by trastuzumab in MCF-7HER2 cells treated 48 h with 1 μM lapatinib compared with untreated cells. The experiment was repeated three times. Student's t-test: *P<0.05.

Discussion

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.

Figure 7
figure7

Proposed alternative mechanism of action of lapatinib based on HER receptor accumulation. In undisturbed conditions, upon ligand binding, the HER receptors form dimers and are phosphorylated (P) by their kinase domains (K). Once phosphorylated, HER2 dimers initiate signaling and undergo ubiquitination (Ub) and lysosomal degradation. Trastuzumab promotes receptor ubiquitination and degradation as well. Lapatinib counteracts receptor phosphorylation, ubiquitination and degradation resulting in HER2 dimer accumulation at the plasma membrane and rendering the cells more susceptible to the immune-mediated action of the anti-HER antibodies (mAbs).

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.

Ubiquitination assay

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.

Immunohistochemistry

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.

Statistical Analysis

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.

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Acknowledgements

This work was supported in full by a grant of the Breast Cancer Research Foundation.

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Correspondence to J Baselga.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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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

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Keywords

  • ErbB receptor
  • trastuzumab
  • tyrosine kinase inhibitor
  • antibody-dependent cell cytotoxicity (ADCC)
  • breast cancer

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