Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models

Lapatinib and saracatinib were purchased from Selleck Chemicals, Munich, Germany. Cetuximab was provided by ImClone Systems NJ, USA. Human breast cancer cell lines MDA-MB-361, SKBR-3, and BT474 were obtained from the American Type Culture Collection. The KPL4 cell line was isolated from the malignant pleural effusion of a breast cancer patient with an inflammatory skin metastasis; these cells are resistant to trastuzumab in female athymic nude mice [22]. The JIMT-1 cell line was established from a pleural metastasis of a 62-year-old breast cancer patient who was clinically resistant to trastuzumab.

Ethical approval by the local ethical committees and patient consent were obtained for the use of KPL4 and JIMT-1 cells. Ethical approval was obtained from the University of Tampere and Tampere University Hospital, Finland, for the JIMT-1 cells. Use of the KPL4 cells was approved by the Kawasaki Medical School, Kurashiki, Okayama, Japan. JIMT-1 cells form trastuzumab-resistant xenograft tumors in nude mice [23]. MDA-MB-361 lapatinib-resistant (LR) cells were generated by using a validated protocol of in vivo/in vitro selection after prolonged exposure to the drug [24]. All cell lines were authenticated by using DNA fingerprinting and maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 20 mM HEPES, pH 7.4, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 4 mM glutamine (ICN, Irvine, UK) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.

Cells (10⁴ cells/well) were grown in 24-well plates and exposed for 72 hours to increasing doses of lapatinib, saracatinib, cetuximab, or their combinations. The percentage of cell survival was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). The half-maximal inhibitory concentration (IC₅₀) for each drug was calculated by GraphPad Prism 5.0 software, normalizing the response between 0 and 100%.

Cancer cell-line monolayers grown to confluence on gridded plastic dishes were wounded by scratching them with a 200-μl pipette tip, and then grown in the presence or absence of each drug (saracatinib, lapatinib, or cetuximab) alone or in combinations. The wounds were photographed (10× objective) at 0 and 24 hours, and healing was quantified by measuring the distance between the edges of the wound by using Adobe Photoshop (v. 8.0.1; Adobe Systems, Inc., San Jose, CA, USA). The results are reported as the percentage of the total distance of the original wound enclosed by cells.

The invasive potential of cancer cells was determined by using a model based on co-culture with fibroblasts, as previously described [25]. In brief, fibroblasts were seeded (10⁴ cells/well) in 24-well plates. After confluence, cells were permeabilized with dimethyl sulfoxide (DMSO; 500 μl) and subsequently overlaid with tumor cells. One hour later, the individual wells were treated with saracatinib, lapatinib, or cetuximab, or their combinations. Eighteen hours later, cells were incubated for 15 minutes with 0.2% trypan blue/phosphate-buffered saline (Mediatech, Herndon, VA, USA). To measure invasion, cells were lysed with 100 μl of 1% sodium dodecyl sulfate/phosphate-buffered saline. Absorbance was measured with a microplate Synergy HT-Bioteck at 610 nm and compared with the absorbance of fibroblasts not overlaid with tumor cells. The results were expressed as percentage of invasion of the fibroblast monolayer with the following formula: X = 100% (cell line and fibroblast well absorbance/fibroblast well absorbance) [25].

Total cell lysates from cell cultures or tumor specimens were resolved by 4% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-human antibodies (see Additional file 1). Co-immunoprecipitation analyses were performed with anti-Src, anti-HER2, or anti-EGFR antibodies; membranes were blotted with anti-Src, anti-HER2, anti-EGFR, or anti-HER3 antibodies. The total lysate from BT474 cells served as positive control. Immunoreactive proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, IL, USA). Densitometry was performed with Image J software (NIH, Bethesda, MD, USA). Details of the complete procedure are reported in Additional file 1.

Small interfering RNA (siRNA) against EGFR and Src were obtained from Invitrogen Life Technologies and Ambion Life Technology (Grand Island, NY, USA), respectively. A nonsense sequence was used as negative control. Details of the complete procedure are reported in Additional file 1.

Five-week-old Balb/c athymic (nu+/nu+) mice (Charles River Laboratories, Milan, Italy), maintained in accordance with the guidelines of the University of Naples “Federico II” Animal Care Committee (ethical approval protocol number 65), were injected orthotopically in the fourth mammary fat pad, with JIMT-1 cells (10⁷ cells/mouse) resuspended in 200 μl of Matrigel (CBP, Bedford, MA, USA). Seven days later, when tumors became detectable, mice (10/group) were randomized to receive lapatinib, 100 mg/kg intraperitoneally (i.p.) 5 times per week for 3 weeks; saracatinib, 50 mg/kg via oral gavage 5 times per week for 3 weeks; cetuximab, 10 mg/kg i.p. twice a week for 3 weeks, or their combinations. Animals treated with dimethyl sulfoxide (DMSO) vehicle served as controls. Tumor volume (cm³) was measured by using the formula π/6 × largest diameter × (smallest diameter)²[26].

Before inoculation with JIMT-1 cells, mice (six mice/group) were treated with lapatinib (100 mg/kg, i.p.), saracatinib (50 mg/kg per os) or both for a week. Then 30 × 10⁵ cells were injected into each animal’s tail vein, after which mice were treated with lapatinib, saracatinib, or both for 7 consecutive days. All mice were killed 21 days after the injection of tumor cells [27]. Human DNA in all lobes of the lungs of the mice was measured by quantifying Alu sequences by polymerase chain reaction, as described elsewhere [28] and detailed in Additional file 1.

The results of in vitro experiments were analyzed with Student t test and expressed as means and standard deviations (SDs) for at least three independent experiments performed in triplicate. The statistical significance of tumor growth was determined by one-way ANOVA and the Dunnett multiple comparison posttest, whereas the log-rank test was used to determine the statistical significance of mouse survival. All reported P values were two-sided. Analyses were performed with the BMDP New System statistical package version 1.0 for Microsoft Windows (BMDP Statistical Software, Los Angeles, CA, USA).

We first evaluated the effect of lapatinib in a panel of HER2-positive cells: MDA-MB-361, SKBR-3, BT474, KPL4, and JIMT-1. As shown in Figure 1A and in Additional file 2: Figure 1A, lapatinib was very active against SKBR-3 (IC₅₀ = 0.11 μM), BT474 (IC₅₀ = 0.06 μM) and MDA-MB361 (IC₅₀ = 0.18 μM); KPL-4 cells were moderately sensitive (IC₅₀ = 0.47 μM) and JIMT-1 cells were resistant (IC₅₀ = 1.9 μM). MDA-MB-361 cells have been reported to be lapatinib resistant in two studies [29, 30] and lapatinib sensitive in another study [18]. In our hands, MDA-MB-361 cells were efficiently inhibited by lapatinib both in vitro and in vivo (data not shown). We also generated an MDA-MB-361 lapatinib-resistant (MDA-MB-361-LR) cell line from parental MDA-MB-361 cells by using a validated protocol of in vivo/in vitro selection [24] after prolonged exposure to the drug. In these derivative lapatinib-resistant cells, the IC₅₀ was reached at the dose of 1.1 μM (Figure 1A and Additional file 2: Figure 1A).

We also tested the effects of the Src inhibitor saracatinib on all the previously described cell lines. Saracatinib alone marginally affected survival of all tested breast cancer cells (IC₅₀ ranging between 0.8 and 2.0 μM), whereas the saracatinib/lapatinib combination exerted a statistically significant inhibitory effect in lapatinib-resistant cells (Figure 1A and Additional file 3: Table S1). In the attempt to shed light on the interaction and the possible synergism between saracatinib and lapatinib, we measured the combination index (CI) devised by Chou and Talalay by using an automated calculation software (CalcuSyn Soft.) [31]. A combination is deemed synergistic when the CI is <1.0, and highly synergistic when the CI is <0.5. As shown in Additional file 2: Figure S1B, the saracatinib/lapatinib combination was highly synergistic in KPL4 cells (median CI, 0.046 ± 0.022), and in lapatinib-resistant cell lines (median CI, 0.21 ± 0.089 for MDA-MB-361-LR, 0.37 ± 0.084 for JIMT-1). The saracatinib/lapatinib combination was not synergistic in MDA-MB-361, SKBR-3, or BT474 cells (data not shown). Details of the combination effect calculation are reported in Additional file 1.Our study of the activation of HER2-dependent signaling in breast cancer cells demonstrated increased levels of Src phosphorylation at tyrosine 416 (Y416) in the lapatinib-resistant cells (MDA-MB-361-LR and JIMT-1), whereas levels of phosphorylated HER2 were unchanged (Figure 1B). Therefore, we examined the effects of saracatinib, lapatinib, and their combination on HER-dependent signal transduction. Saracatinib slightly inhibited HER2-related transducers in MDA-MB-361, SKBR-3, MDA-MB-361-LR, and JIMT-1 cells. It efficiently reduced Src activity in all these cells (Figure 1C). Lapatinib reduced HER2, Akt, and MAPK phosphorylation/activation in the lapatinib-sensitive MDA-MB-361 and SKBR-3 cells. It also reduced HER2 phosphorylation in MDA-MB-361-LR and JIMT-1 cells (about 50% in both cell lines), but its effect on Akt and MAPK phosphorylation was significantly lower in resistant cells than in sensitive MDA-MB-361 and SKBR-3 cells. In all cell lines, inhibition of signal transduction was greater in cells treated with the saracatinib/lapatinib combination than in cells treated with a single agent. It almost completely suppressed pHER2, pAkt, pSrc, and pMAPK levels (Figure 1C).

Because Src activation is a well-known driver of tumor metastasis, we investigated the effects of its inhibition on the migration and invasive potential of breast cancer cells sensitive or resistant to lapatinib. In the absence of drugs, resistant cells were more aggressive than sensitive cells, as witnessed by their higher migration (Figure 2A) and invasion (Figure 2B). Treatment with low doses of saracatinib inhibited migration of all human breast cancer cell lines in a wound-healing assay, whereas lapatinib did not exert any effect (Figure 2C). The combination of saracatinib and lapatinib strongly inhibited migration in the lapatinib-resistant MDA-MB-361-LR and JIMT-1 cells, whereas this effect was less pronounced in the lapatinib-sensitive MDA-MB-361 and SKBR-3 cells (Figure 2C and Additional file 4: Table S2). In addition, whereas neither saracatinib nor lapatinib alone significantly affected invasion of a fibroblast monolayer by breast cancer cells, the combined treatment did, even in lapatinib-resistant cells. The results were less evident in SKBR-3 cells, because of their low-motility capabilities (Figure 2D and Additional file 4: Table S2); for the same reason, BT474 and KPL4 cells were not included in the analysis.We next analyzed the effect of the saracatinib/lapatinib combination on signal transducers involved in cell migration and invasion (Figure 2E). Whereas saracatinib treatment decreased pFak, ppaxillin, and pp130Cas levels in MDA-MB-361-LR and JIMT-1 cell lines, lapatinib alone was less effective. Notably, saracatinib plus lapatinib treatment resulted in reduction of all the previously cited mediators, which is in line with their inhibitory effects on migration and invasion processes (Figure 2E).

To test the efficacy of the saracatinib/lapatinib combination and of each agent given alone in vivo, we orthotopically xenografted the lapatinib-resistant JIMT-1 cells in Balb/C nude mice, and measured both tumor growth and survival. As shown in Figure 3A, in untreated mice, the tumor reached the maximum allowed size (about 2 cc, on day 63, which was 9 weeks after the injection of tumor cells. At this time, saracatinib and lapatinib inhibited tumor growth by 37% and 13%, respectively, whereas the saracatinib/lapatinib combination inhibited tumor growth by 75%. In lapatinib-treated mice, tumors reached 2 cc on day 77, 10 weeks after tumor injection versus day 98 (14 weeks after tumor injection) in saracatinib-treated mice. Saracatinib plus lapatinib synergistically exerted a potent long-lasting antitumor effect: 42% growth inhibition (tumor size, 1.16 cm³) at the end of the experiment (day 105). At ANOVA test, tumors were significantly larger in mice treated with a single agent than in combination-treated mice (combination versus single agents, P < 0.1 × 10^-2 at the median survival of the control group) (Figure 3A). Consistently, median survival was significantly longer in mice treated with the saracatinib/lapatinib combination than in both control mice (median survival, 102.50 versus 32.50 days; hazard ratio, 0.03751; 95% confidence interval, 0.009463 to 0.1487; P < 0.1 × 10^-3) and in mice treated with saracatinib alone (median survival, 102.50 versus 61.50 days; hazard ratio, 0.1785; 95% confidence interval, 0.05776 to 0.5518; P = 0.28 × 10^-2) (Figure 3B). Treatments were well tolerated; no weight loss or other signs of acute or delayed toxicity were observed. No spontaneous macrometastases were found in brains, lungs, spleens, or livers after death in any of the mouse groups.

To investigate the effect of saracatinib and lapatinib on tumor metastatic behavior, we injected JIMT-1 cells into the tail vein of Balb/c nude mice and then treated the animals with saracatinib, lapatinib, or both. Examination of serial histologic sections of mouse lungs did not reveal macrometastases (data not shown). To identify micrometastases in the lung, we measured human DNA in mice lungs by using real-time polymerase chain reaction (PCR) for human Alu sequences, as previously described [28]. Human DNA was detected in the lungs of untreated mice (Additional file 5: Figure S2). Saracatinib was much more effective than lapatinib in reducing levels of human DNA in mouse lungs. However, as shown in Figure 3C, combined treatment inhibited the formation of micrometastases by 90% (combination versus saracatinib, P < 0.1 × 10^-2).Western-blotting analysis of tumors removed on completion of treatment revealed that saracatinib reduced the activated forms of HER2, EGFR, Akt, MAPK, Src, FAK, paxillin, and p130Cas (Crk-associated substrate). It also increased the inactive form of Src phosphorylated on tyrosine 527 (Y527). As expected, lapatinib did not inhibit HER-dependent signaling in JIMT-1 cells. The combination of saracatinib and lapatinib inhibited signal transduction more effectively than did single-agent treatments; it almost completely suppressed the activated forms of HER2, Akt, MAPK, Src, paxillin, and p130Cas (Figure 3D).

Given the antitumor effects induced by saracatinib treatment on lapatinib-resistant cells, in vitro and in vivo, we used siRNA against Src to exclude off-target effects of saracatanib. We found that Src silencing reduced Src levels by about 40% in MDA-MB-361-LR cells (P = 1.9 × 10^-2) and by 48% in JIMT-1 cells (P = 2 × 10^-2) (Figure 4A). Similar levels of Src reduction were observed in lapatinib-sensitive MDA-MB-361 and SKBR-3 cells (>80% in both cases). In resistant cell lines, Src silencing moderately inhibited EGFR-related transducers, but efficiently reduced Src activation. Moreover, coupled with lapatinib, it greatly reduced Src, Akt, and MAPK activation, thereby recapitulating the effects observed with saracatinib plus lapatinib. Conversely, in sensitive cells, the combined treatment was not more effective than lapatinib alone in inhibiting HER2-dependent signal transduction (Figure 4A).

Scr family kinases interact with various transmembrane receptors, including members of the HER family [20, 32], and regulate several cellular activities. Therefore, we investigated whether Src participates in HER signaling in lapatinib-resistant cells by interacting with RTKs. As shown in Figure 4B, in co-immunoprecipitation experiments, the HER2/Src association was much lower in the lapatinib-resistant cells MDA-MB-361-LR and JIMT-1 than in the lapatinib-sensitive MDA-MB-361 and SKBR-3 cells. Conversely, the interaction between EGFR and Src was much more evident in MDA-MB-361-LR and JIMT-1 than in sensitive cells. HER3 did not co-immunoprecipitate with Src. Moreover, the level of HER2/EGFR heterodimers was greatly reduced, and no HER3/EGFR heterodimers were found in lapatinib-resistant cells (Figure 4B). To probe the molecular mechanism underlying EGFR activation, we performed ELISA assays for the EGFR ligands EGF and transforming growth factor (TGF)-α, and found no significant difference in the expression/secretion of EGFR ligands among the various lapatinib-sensitive and lapatinib-resistant cell lines (see Additional file 6: Figure S3A, B). Details of the complete procedure are reported in Additional file 1.

A site of tyrosine phosphorylation has been identified within the activation loop of the EGFR TK domain, tyrosine 845 (Y845), whose phosphorylation is mediated by Src [33‐35]. Consistent with the enhanced levels of Src activation and with the EGFR/Src interaction detected in lapatinib-resistant cells, EGFR phosphorylation on Y845 was higher in the lapatinib-resistant MDA-MB361-LR and JIMT-1 cells than in the lapatinib-sensitive cells, whereas Y1173 phosphorylation did not differ between lapatinib-resistant and lapatinib-sensitive cells (Figure 4C).

To investigate the role of EGFR in Src-mediated lapatinib resistance, we evaluated the effect of the anti-EGFR mAb cetuximab combined with lapatinib on signal transduction of lapatinib-resistant breast cancer cells. We found that cetuximab but not lapatinib reduced EGFR phosphorylation on Tyr1173 (Figure 5A). Interestingly, cetuximab inhibited Src-mediated phosphorylation/activation of EGFR by reducing levels of phospho-Y845. Consistently, cetuximab alone reduced Src phosphorylation in MDA-MB361-LR cells and abolished it in JIMT-1 cells.

A reduction of phospho-Akt and phospho-MAPK levels was also observed, and this effect was even more evident with the addition of lapatinib to cetuximab compared with cetuximab alone. Conversely, in lapatinib-sensitive cells, the lapatinib/cetuximab combination was not more effective than lapatinib alone in inhibiting signal transduction (Figure 5A). Moreover, an analysis of Src-related effectors of cellular motility showed that combination treatment induced a reduction of the active forms of FAK, paxillin, and p130Cas (see Additional file 7: Figure S4A).

We next used an EGFR-specific siRNA to verify the role of EGFR in Src activation in lapatinib-resistant cells, and found that it reduced EGFR expression levels by about 43% in MDA-MB-361-LR cells (P = 0.6 × 10^-2) and by 68% in JIMT-1 cells (P = 0.4 × 10^-2) (Figure 5B). This effect was paralleled by a reduction of pSrc levels. Moreover, as observed after cetuximab treatment, levels of the phosphorylated forms of Akt and MAPK were reduced after treatment with EGFR siRNA, alone or associated with lapatinib. Conversely, in lapatinib-sensitive cells, combined treatment was not more effective than lapatinib alone in inhibiting signal transduction (Figure 5B).

To assess the role of Src-dependent EGFR activation in the onset of lapatinib resistance, we transfected the sensitive MDA-MB-361 cell line with either the wild-type EGFR or the mutant Tyr845Phe (Y845F) variant of EGFR. As shown in Additional file 7: Figure S4B, overexpression of wild-type EGFR drastically reduced the inhibition of proliferation induced by lapatinib treatment, an effect partially rescued by the mutant Y845F, which also demonstrates the functional interaction between Src and EGFR.

Given that the combination of cetuximab and lapatinib efficiently blocked signal transduction, we investigated whether this approach could overcome lapatinib resistance in MDA-MB-361-LR and JIMT-1 cells. In the parental MDA-MB-361 cell line, lapatinib controlled cell survival; the addition of cetuximab did not increase this effect (Additional file 8: Figure S5A and Additional file 9: Table S3). The medium CI, measured with the Chou and Talalay method [31] and using a constant dose ratio (2.8:1), was 1.189 ± 2.7 (Additional file 8: Figure S5B).

In the lapatinib-resistant MDA-MB-361-LR and JIMT-1 cells, treatment with lapatinib or cetuximab alone did not affect cancer cell survival, whereas the combination of the two drugs at an equipotent ratio greatly reduced it (Additional file 8: Figure S5A and Table S3). As shown in Additional file 8: Figure S5B, the synergistic effect of combination treatment was very strong (median CI, 0.029 ± 1.83 for MDA-MB-361-LR cells, and 0.204 ± 5.74 for JIMT-1 cells). Conversely, the combination of cetuximab plus saracatinib was not more effective than each single agent (see Additional file 10: Figure S6A). Interestingly, cetuximab partially reduced migration and invasion of lapatinib-resistant cells. The addition of cetuximab to lapatinib efficiently blocked the migration and invasion capabilities of resistant cells (Additional file 8: Figure S5C, D, and Additional file 11: Table S4), as observed with the saracatinib-plus-lapatinib combination.

We tested the effect of cetuximab combined with lapatinib also in vivo, in Balb/C nude mice orthotopically xenografted with the lapatinib-resistant JIMT-1 cells. When tumors in untreated mice reached the maximum allowed size, about 2 cc, which occurred on day 49, cetuximab and lapatinib, given singly, inhibited tumor growth by 49% and 15%, respectively. The two agents combined reduced tumor growth by 88%. In none of the treated animals did the tumor size reach 2 cc on day 49, 7 weeks after tumor injection. One-way ANOVA revealed a significantly longer median survival in mice treated with the combination than in those treated with single agents (P < 0.1 × 10^-2) evaluated at the median survival period of the control group (Figure 6A). We were unable to calculate the median survival of mice treated with the combination because 90% of the mice were alive on day 49 (Figure 6B). Treatments were well tolerated; no weight loss or other signs of acute or delayed toxicity were observed.

As shown in Figure 6C, Western blotting analysis of tumors removed at the end of treatment revealed that, as expected, lapatinib did not inhibit HER-dependent signaling. Cetuximab reduced the activated forms of EGFR, MAPK, and Src. The effect on the EGFR probably reflects a reduction of the total EGFR expression due to cetuximab-induced internalization and degradation [36]. The cetuximab/lapatinib combination was even more effective in inhibiting signal transduction than was each agent administered alone (Figure 6C).