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Cancer Biology and Signal Transduction

Resistance Mechanism against Trastuzumab in HER2-Positive Cancer Cells and Its Negation by Src Inhibition

Mei Hua Jin, Ah-Rong Nam, Ji Eun Park, Ju-Hee Bang, Yung-Jue Bang and Do-Youn Oh
DOI: 10.1158/1535-7163.MCT-16-0669 Published June 2017

Abstract

Trastuzumab in combination with chemotherapy is the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-positive breast and gastric cancers. Several resistance mechanisms against anti-HER2 therapy have been proposed. Src activation has been suggested to be responsible for the resistance of HER2-positive breast cancer. In our study, we generated four trastuzumab-resistant (HR) cancer cell lines from HER2-amplified gastric and biliary tract cancer cell lines (SNU-216, NCI-N87, SNU-2670, and SNU-2773). Elevated Src phosphorylation was detected in SNU2670HR and NCI-N87HR cell lines, but not in SNU216HR or SNU2773HR cell lines. In SNU216HR and SNU2773HR cell lines, phospho-FAK (focal adhesion kinase) was elevated. Bosutinib as a Src inhibitor suppressed growth, cell-cycle progression, and migration in both parental and HR cell lines. Specifically, Src interacted with FAK to affect downstream molecules such as AKT, ERK, and STAT3. Bosutinib showed more potent antitumor effects in Src-activated HR cell lines than parental cell lines. Taken together, this study suggests that Src inhibition may be an effective measure to overcome trastuzumab resistance in HER2-positive cancer. Mol Cancer Ther; 16(6); 1145–54. ©2017 AACR.

Introduction

Human epidermal growth factor receptor 2 (HER2; also known as ERBB2) is overexpressed or amplified in approximately 15% to 20% of breast cancers, 10% to 15% of gastric cancers, and 10% to 20% of biliary tract cancers (1–3). Trastuzumab is a humanized monoclonal antibody that binds to the extracellular juxtamembrane domain of HER2 and blocks intracellular tyrosine kinase activation (4). Currently, trastuzumab plus chemotherapy is the standard of care for patients with HER2-positive breast and gastric cancers. However, resistance to anti-HER2 therapy eventually develops as acquired or primary resistance (4). Multiple resistance mechanisms against anti-HER2 therapy have been reported. The HER2 expression level is mainly considered as a biomarker for patient selection in clinical practice (5).

Src is a proto-oncogene and coordinates multiple signaling pathways involved in tumor progression (6, 7). High levels of pSrc expression have been found in various cancers, such as colon, liver, lung, breast, and pancreatic cancers (7). Src can be activated by many factors, including receptor tyrosine kinases (RTK), G protein–coupled receptors, adhesion receptors, and cytokine receptors (7). Src has two major phosphorylation sites, tyr416 and tyr527. When Src is activated, tyr416 is phosphorylated and tyr527 is dephosphorylated (8, 9). Upon Src activation, it can upregulate multiple downstream molecules, such as AKT, ERK, and STAT3, leading to increases in cell proliferation and survival (10). Extensive preclinical evidence indicates that Src activation facilitates cell motility, and that Src participates in stimulation of angiogenesis under hypoxic conditions (9). Moreover, Src interacts with focal adhesion kinase (FAK) to accelerate cell migration, invasion, and cell-cycle progression (9, 11–12). Promotion of metastasis is one of the most principal roles of Src activation (13). Additionally, Src enhances E-cadherin and integrin signaling that may activate various other signaling networks (14).

Several studies have suggested that Src contributes to trastuzumab resistance in HER2-positive breast cancer (9, 15–19). The mechanisms of Src activation in trastuzumab-resistant (HR) breast cancer could be phosphatase and tensin homolog (PTEN) mutation that directly induces Src activation (9). CUB domain-containing protein 1 interacts with HER2 and promotes Src activation (18). Furthermore, transforming growth factor-β binds to HER2 and integrins, leading to Src activation (19). In breast cancer, in addition to trastuzumab resistance, Src activation has a crucial role in resistance to lapatinib (HER1 and HER2 dual inhibitor; ref. 20). In HER2-positive gastric cancer for which trastuzumab is the standard of care, the role of Src in trastuzumab resistance has not been studied. Recently, we have suggested that HER2 is a promising therapeutic target in biliary tract cancer, similar to breast and gastric cancers (21).

This study was conducted to uncover the role of Src in trastuzumab resistance and evaluate the effect of Src inhibition in overcoming trastuzumab resistance of HER2-positive gastric and biliary tract cancer cells.

Materials and Methods

Cell lines and reagents

We established two patient-derived, HER2-amplified biliary tract cancer cell lines, SNU2670 and SNU2773, in September 2013 and November 2014, respectively (21). SNU216 and NCI-N87, two HER2-amplified gastric cancer cell lines, were purchased from the Korean Cell Line Bank (Seoul, South Korea) in May 2013. The most recent authentications of the four cell lines were performed using an AmpFLSTR Identifiler PCR Amplification Kit (catalog no. 4322288; Applied Biosystems) by the Korean Cell Line Bank on December 14, 2016, and January 10, 2017. A 3530xl DNA Analyzer (Applied Biosystems) and GeneMapper v5 (Applied Biosystems) were used for DNA finger-printing analysis. All cells were cultured in RPMI-1640 medium (Welgene Inc., Gyeongsan, Korea) containing 10% fetal bovine serum and 10 μg/mL gentamicin at 37°C with 5% CO2. Trastuzumab was purchased from Roche (Korea). Bosutinib (4-anilino-3-quinolinecarbonitrile; SKI-606), a dual Src/ Bcr-Abl kinase inhibitor was purchased from Selleck Chemicals LLC for in vitro experiments and provided by Pfizer Inc. for in vivo experiments.

Generation of trastuzumab-resistant (HR) cell lines

HR cell lines were established using four HER2-amplified cell lines. SNU216HR, NCI-N87HR, SNU2670HR, and SNU2773HR cell lines were derived from the parental cell lines and maintained in medium containing 20 or 50 μg/mL trastuzumab. After approximately 1 year in culture with trastuzumab, HR cell lines were established.

Cell growth inhibition assay

Cells were seeded in 96-well plates at density of 0.8–8×103 cells per well and incubated overnight at 37°C. The cells were then exposed to increasing concentrations of bosutinib alone or in combination with trastuzumab for 72 hours. Then, 50 μL 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (Sigma Aldrich) was added to each well, followed by incubation at 37°C. Four hours later, the medium was removed and 150 μL dimethyl sulfoxide was added to each well. After 5 minutes, the absorbance of each well was measured at 540 nm with a VersaMax Microplate Reader (Molecular Devices). The half-maximal inhibitory concentration (IC50) of agents was analyzed using SigmaPlot software (Systat Software, Inc.). The experiments were performed in triplicate.

Colony formation assay

Cells (0.5–4×103) were seeded in 6-well plates and exposed in various concentrations of trastuzumab or bosutinib. After 10 to 16 days, colonies were stained with Coomassie blue for 2 hours and then counted using Gel doc system software (Bio-Rad). The half-maximal inhibitory concentration (IC50) of agents was analyzed using SigmaPlot software (Systat Software, Inc.). Each experiment was repeated three times.

Western blotting

Cells (3–8×105) were seeded in 100-mm dishes and treated with trastuzumab (10 μg/mL), bosutinib (0.01, 0.1, and 1 μmol/L), or trastuzumab (10 μg/mL) plus bosutinib (0.01, 0.1, and 1 μmol/L) for 24 hours. Then, the cells were harvested and lysed in RIPA buffer containing protease inhibitors on ice for 30 minutes. Next, the proteins were recovered by centrifugation at 13000 rpm for 20 minutes. Primary antibodies against the following molecules were purchased from Cell Signaling Technology: pEGFR-Tyr1068 (#3777), EGFR (#2232), pHER2-Tyr1248 (#2247), HER2 (#2242), pHER3-Tyr1289 (#4791), HER3 (#12708), pSrc-Tyr416 (#2101), Src (#2108), pAKT-Ser473 (#9271), AKT (#9272), pFAK-Tyr925 (#3284), FAK (#3285), pSTAT3-Tyr705 (#9131), STAT3 (#12640), pERK-Thr202/Tyr204 (#9101), ERK (#9102), and PTEN (#9559). An anti-E-cadherin (#610181) antibody was purchased from BD Bioscience, and an anti–β-actin antibody was purchased from Sigma-Aldrich. Secondary antibodies were acquired from Thermo Scientific Inc.

Cell-cycle analysis

Cells (0.5–2.5×105) were seeded in 60-mm dishes and treated with various concentrations of bosutinib (0.01, 0.1, and 1 μmol/L) for 24, 48, and 72 hours. Then, the cells were harvested and fixed with 70% ethanol at −20°C. After 2 days, 7 μL RNAse A (Invitrogen; 20 mg/mL) was added to each well, followed by incubation for 10 minutes at 37°C. The cells were then stained with 13 μL propidium iodide (Sigma-Aldrich) and analyzed by a FACSCalibur flow cytometer (BD Bioscience). Each experiment was repeated three times.

Migration assay

Cells (3–8 × 105) were seeded in 6-well plates and incubated at 37°C. After 24 hours, the cells were scratched with a 200-μL pipet tip and treated with various concentrations of bosutinib (0.01, 0.1, 1, and 10 μmol/L), trastuzumab (0.01, 0.1, 1, 10, and 100 μg/mL), or in combination. Images were analyzed using ImageJ software at 0, 6, 24, 48, and 72 hours. All experiments were performed in triplicate.

In vivo experiments

Animal experiments were performed at the Biomedical Center for Animal Resource Development of Seoul National University (Seoul, South Korea) according to the institutional guidelines with prior approval from the institutional animal care and use committee. Four-week-old female athymic nude mice were purchased from Central Lab Animal Inc. (Seoul, South Korea). Each mouse was injected subcutaneously with 2 × 107 SNU2670 or SNU2670HR cells. When the tumor volume reached 200 mm3, the mice were divided into four groups (four mice per group). Bosutinib was administered orally once a day at 150 mg/kg for 3 weeks. Control groups were treated with the vehicle (0.5% methylcellulose and 0.4% Tween 80) through oral gavage. Body weights and tumor size were measured every other day. The tumor volume was calculated by the formula: tumor volume = [(width)2 × height]/2.

Immunohistochemistry (IHC)

Sections of paraffin-embedded tumor tissues were deparaffinized and dehydrated. IHC detection of proliferating cells was conducted using an anti–Ki-67 antibody (GeneTex, Inc.) at a dilution of 1:100. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were conducted to detect apoptosis using an ApopTag In situ Apoptosis Detection Kit (EMD Millipore) according to the manufacturer's protocol.

Statistical analysis

Statistical analyses were conducted using SigmaPlot version 9.0. Experimental data are presented as the mean ± standard error or deviation. All statistical tests were two-sided. Differences were considered statistically significant at P < 0.05. In MTT assays, P values were calculated by comparing parental and HR cell viabilities at each trastuzumab concentration. In the in vivo xenograft experiments, P values were calculated by comparing tumor volumes at each treatment day.

Results

Characterization of HR cell lines

First, to identify whether HR cell lines were successfully established, eight cancer cell lines were treated with trastuzumab. In MTT assays, the growth of four HR cell lines (SNU216HR, NCI-N87HR, SNU2670HR, and SNU2773HR) was not inhibited by trastuzumab at up to 100μg/mL (P < 0.05; Fig. 1A). Next, we compared the expression of signaling molecules between parental and HR cell lines (Fig. 1B). We found that the four HR cell lines could be subdivided into two groups according to the Src activation level. In SNU2670 and NCI-N87 cell lines that expressed pSrc weakly, pSrc was activated upon development of trastuzumab resistance (SNU2670HR and NCI-N87HR cell lines). Conversely, in SNU216 and SNU2773 cell lines that expressed high levels of pSrc, the level of pSrc was decreased when they developed trastuzumab resistance (SNU216HR and SNU2773HR cell lines). In these cell lines, phosphorylation of FAK was elevated. However, no change in the levels of total Src was observed in all four HR cell lines. In breast cancer, HR cell lines exhibit lower HER2 expression than parental cell lines (9). In our study, two biliary tract cancer HR cell lines (SNU2773HR and SNU2670HR), but not gastric cancer HR cell lines (SNU216HR and NCI-N87HR), showed decreased HER2 levels compared with the parental cell lines. We also found that pEGFR was prominently upregulated in the four HR cell lines compared with the parental cell lines. However, there was no change in PTEN expression levels between parental and HR cell lines (Supplementary Fig. S1).

Figure 1.
Figure 1.

Characterization of trastuzumab-resistant (HR) cell lines. A, Both parental and HR cell lines were treated with increasing concentrations of trastuzumab for 72 hours. Cell viability was measured by MTT assays. *, P < 0.05. B, Changes in basal expression levels of molecules involved in signaling pathways of HR cell lines compared with parental cell lines.

Bosutinib inhibits the growth of both parental and HR cell lines, especially Src-activated HR cell lines

To evaluate the antiproliferative effect of bosutinib in HER2-amplified cancer cell lines, we measured cell viabilities following bosutinib treatment for 72 hours by MTT assays. As a result, the proliferation of all eight cell lines was inhibited by treatment with bosutinib alone, and SNU2670HR cells were the most sensitive to bosutinib (Supplementary Fig. S2A; Table 1). Based on the IC50 values, it was clearly observed that Src-activated NCI-N87HR and SNU2670HR cell lines were more sensitive to bosutinib compared with parental cell lines (Table 1). We next used colony formation assays to identify the long-term antiproliferative effects of bosutinib (Supplementary Fig. S2B). Our data indicated that six cell lines showed growth inhibition by bosutinib (Supplementary Fig. S2B; Table 1).

View this table:
Table 1.

IC50 by MTT assay and colony formation assay of bosutinib treatment

These data suggested that bosutinib had antiproliferative effects in both parental HER2-amplified cell lines and HR cell lines, especially Src-activated cell lines such as SNU2670HR and NCI-N87HR.

Src contributes to trastuzumab resistance and an Src inhibitor downregulates the signaling pathway

To validate the effect of bosutinib on the HER2 signaling pathway, we detected Src-related molecules after treatment with bosutinib for 24 hours. Bosutinib inhibited Src pathway signals in both parental and HR cell lines (Fig. 2). In Src-activated HR cell lines (NCI-N87HR and SNU2670HR), pAKT, pERK, and pSTAT3 were more potently inhibited by bosutinib than in parental cell lines. In SNU216HR and SNU2773HR cell lines, although pAKT and pERK were less inhibited by bosutinib compared with parental cell lines, Src-dependent FAK phosphorylation, which is involved in cell-cycle progression and migration, was downregulated significantly.

Figure 2.
Figure 2.

Src inhibitor downregulates the related signaling pathway. Both parental and HR cell lines were treated with bosutinib (0.01, 0.1, and 1 μmol/L) for 24 hours. Src-related signaling pathway molecules were then analyzed.

These results supported an important role of Src activation that might contribute to trastuzumab resistance.

Bosutinib influences the cell cycle

To evaluate the effect of bosutinib on the cell cycle, the eight cell lines were treated with bosutinib for 24, 48, and 72 hours. As shown in Fig. 3, 1 μmol/L bosutinib induced G1 arrest in SNU216HR and SNU216 cell lines (both P < 0.05). Bosutinib also induced G1 arrest in SNU2773HR cells (P < 0.05) and SNU2773 cells (P = 0.053). These data indicated that, in pSrc-decreased cells, bosutinib resulted in the same degree of G1 arrest in both parental and HR cell lines. In contrast, the sub-G1 fraction was significantly increased in SNU216HR (P < 0.05) and SNU2773HR (P < 0.01) cell lines, but not in SNU216 or SNU2773 cell lines.

Figure 3.
Figure 3.

Bosutinib influences the cell cycle. After treatment with bosutinib (0.01, 0.1, and 1 μmol/L) for 48 and 72 hours, cell-cycle analysis was conducted. Data represent three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

In the pSrc-increased group, G1 arrest was observed in both SNU2670 (P < 0.05) and SNU2670HR (P < 0.01) cell lines after 72 hours, and the sub-G1 fraction was increased in SNU2670HR cells (P < 0.01). In contrast, there was no change in the cell cycle of both NCI-N87 and NCI-N87HR cell lines.

Bosutinib inhibits migration of HR cell lines more effectively compared with parental cell lines

Src plays an important role in cell migration. To evaluate the effect of bosutinib on cell migration, we conducted migration assays. The eight cell lines were treated with bosutinib for 6, 24, 48, and 72 hours. Images were captured after treatment with bosutinib alone for 48 hours (Fig. 4). We found that HR cells showed faster migration than parental cells, and bosutinib exerted a potent antimigratory effect on both parental and HR cell lines. Migration of all cell lines except NCI-N87 was significantly inhibited by 1 μmol/L bosutinib. Our results suggested that bosutinib inhibited cell migration very effectively, and its effects were more obvious in HR cell lines.

Figure 4.
Figure 4.

Bosutinib inhibits migration of HR cell lines more potently compared with parental cell lines. All cell lines were treated with the indicated concentrations of bosutinib for 48 hours. Images were analyzed by ImageJ software. *, P < 0.05; **P < 0.01; ***, P < 0.001.

Effects of the combination of trastuzumab and bosutinib

In the present study, we observed a potent antitumor effect of bosutinib in Src-activated HR cell lines with respect to parental cell lines. Next, we conducted combined treatments with trastuzumab and bosutinib. Cell viabilities were measured following treatment with trastuzumab, bosutinib, or both for 72 hours. No synergistic effects were observed (Supplementary Fig. S3A). In SNU2773HR, NCI-N87HR, and SNU2670HR cell lines, although there was no significant difference between bosutinib alone or in combination compared with trastuzumab alone, bosutinib still effectively inhibited cell viability. Next, colony formation assays were conducted. The data were similar to the MTT assay results (Supplementary Fig. S3B). After all cell lines were exposed to trastuzumab plus bosutinib for 24 hours, Western blotting was conducted. All signals were blocked by combined treatment and 1μmol/L bosutinib alone (Supplementary Fig. S3C). Furthermore, to evaluate the antimigration effect of the combination of trastuzumab and bosutinib, we captured migration images after 48 hours of drug treatment. We observed that SNU216HR, SNU2773HR, and SNU2670HR cell lines had faster migration compared with their respective parental cell lines. At 48 hours of treatment, the gaps were already closed by these HR cell lines compared with the control. Trastuzumab (10 μg/mL) did not inhibit cell migration in any cell line and the combination of trastuzumab and bosutinib had no synergistic effect (Supplementary Fig. S3D).

Bosutinib overcomes trastuzumab resistance in vivo

In vitro data indicated that bosutinib exerted potent effects in both parental and HR cell lines, especially in Src-activated HR cell lines. To show that targeting Src could overcome trastuzumab resistance in vivo, we established xenografts using SNU2670 and SNU2670HR cell lines. Before testing bosutinib, to ensure SNU2670HR tumors were resistant to trastuzumab, SNU2670 and SNU2670HR tumors were treated with 4 mg/kg trastuzumab for 3 weeks. The results indicated that the growth of SNU2670 tumors was inhibited by trastuzumab, and SNU2670HR tumors showed resistance to trastuzumab (Supplementary Fig. S4).

To evaluate the effect of bosutinib in vivo, SNU2670 and SNU2670HR cells were injected into mice. The mice were divided into four groups including two control groups and two bosutinib treatment groups. Statistical analysis was performed to compare tumor volumes between treatments or between xenografts at each treatment day.

During treatment, bosutinib did not influence body weight (Fig. 5A).

Figure 5.
Figure 5.

Inhibition of in vivo tumor growth by bosutinib in SNU2670 and SNU2670HR xenografts. SNU2670 and SNU2670HR cells (2 × 107) were injected into mice, followed by treatment with 150 mg/kg bosutinib for 21 days. A, Changes in mouse body weights during treatment. B, Tumor growth curves of control (vehicle) and bosutinib groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Arrow indicates P < 0.05 comparing SNU2670-bosutinib and SNU2670HR-bosutinib treatment groups. C, Tumors were harvested, and then hematoxylin–eosin staining (400×) and IHC analysis (400×) were conducted. D, Proteins were harvested from isolated tumors for Western blotting.

In SNU2670 parental xenografts, P values between control and bosutinib-treated groups were less than 0.05 at 15 and 22 days after treatment. In SNU2670-HR xenografts, the difference between control- and bosutinib-treated groups was significant (P < 0.05) at all time points except for day 1 after treatment. When we compared bosutinib-treated groups only, SNU2670-HR xenografts showed greater inhibition of tumor growth than SNU2670 parental xenografts at 15 and 17 days after treatment (P < 0.05; Fig. 5B). Based on these results, we concluded that the obvious antitumor effect of bosutinib was observed in both SNU2670 parental xenografts and SNU2670-HR xenografts. Furthermore, the antitumor effect was greater in SNU2670-HR xenografts than in SNU2670 parental xenografts.

We also detected ki-67 expression and performed TUNEL assays in the xenografted tumor tissues after bosutinib treatment (Fig. 5C). Both SNU2670 and SNU2670HR tumors showed decreased ki-67 expression compared with control groups. TUNEL staining as a biomarker of apoptosis was increased in bosutinib treatment groups, which was consistent with the results of the in vitro cell-cycle analysis.

Moreover, proteins were harvested from two different isolated tumor tissues in each group to conduct Western blotting (Fig. 5D). pSrc was upregulated in HR control tumors compared with parental control groups, but bosutinib downregulated pSrc in both parental and HR tumors. pHER2 and pERK were more efficiently blocked in HR tumors. Taken together, these data suggest that bosutinib can overcome trastuzumab resistance in vivo.

Discussion

Many studies have been performed to clarify the resistance mechanism against anti-HER2 agents in HER2-positive breast cancer (20, 22–26). Several reports have suggested that Src activation is among the key factors (8, 9, 17, 20). In our study, the primary purpose was to explore Src activation involved in trastuzumab resistance and use an Src inhibition strategy to overcome trastuzumab resistance, especially in HER2-positive gastric and biliary tract cancers.

We established four HER2-amplified HR gastric and biliary tract cancer cell lines. There were different characteristics among these HR cell lines (Fig. 1B). Two cell lines (SNU2670 and NCI-N87), which expressed pSrc weakly, showed Src activation in parallel with development of trastuzumab resistance (SNU2670HR and NCI-N87HR cell lines). In contrast, the other cell lines (SNU216 and SNU2773) with high expression of pSrc showed downregulation of pSrc and upregulation of pFAK upon development of trastuzumab resistance (SNU216HR and SNU2773HR cell lines). Supporting our data, a recent study reported that 37.8% of HR tumors show Src activation that indicates a poor prognosis of HER2-positive breast cancer (15).

In our study, an Src inhibitor showed a good antitumor activity in all four HR cell lines, but a more potent antitumor activity was observed in Src-activated HR cell lines (SNU2670HR and NCI-N87HR).

Another study revealed Src activation as a key modulator of the trastuzumab resistance mechanism, and that targeting Src can overcome trastuzumab resistance in breast cancer (8). In our study, we also detected an identical response in Src-activated HR cell lines by the Src inhibitor. In MTT assays, Src-activated HR cell lines (SNU2670HR and NCI-N87HR) showed decreased proliferation by bosutinib monotherapy (Table 1). Furthermore, in these Src-activated HR cell lines, not only pSrc, pAKT, and pERK but also pEGFR, pHER2, and pHER3 were downregulated significantly. Moreover, pFAK (tyr925) was blocked efficiently, which has been identified as an Src-dependent residue. Because of the role of pFAK (tyr925) in cell-cycle progression and migration, we could explain why Src-decreased HR cells also exhibited G1 arrest by bosutinib treatment (Fig. 3).

Among several other studies on blocking Src to overcome anti-HER2 resistance, few have reported the effect of blocking Src on the cell cycle. One study demonstrated that Src inhibition combined with trastuzumab enhances apoptosis of HR breast cancer cells (8). Another study showed that Src inhibition results in only a modest increment of apoptosis (20). Our study revealed that bosutinib monotherapy induced apoptosis of HR cell lines in vitro. In xenograft models, an increase of TUNEL staining was also observed by bosutinib treatment.

One of the well-known functions of activated Src is enhancement of metastasis. Indeed, through migration assays, we observed the promising antimigration effect of bosutinib in HR cell lines (Fig. 4). Bosutinib dramatically impeded HR cell migration compared with parental cell lines. In the in vivo experiments, bosutinib significantly inhibited tumor growth, but more potent inhibition was observed in HR tumors (Fig. 5).

Thus far, a deregulated HER2–AKT pathway is the most well-known mechanism in trastuzumab resistance of HER2-positive breast cancer (23–25). It is known that PTEN loss directly leads to Src activation, but our four HR cell lines did not show any change in PTEN expression levels compared with parental cell lines (Supplementary Fig. S1). PIK3CA mutation or pAKT upregulation have also been considered as the main alterations in HR cells. In our study, we found that two Src-decreased HR cell lines (SNU216HR and SNU2773HR) showed pAKT upregulation. Furthermore, SNU2670HR and NCI-N87HR cell lines exhibited pAKT downregulation. There are conflicting data on pAKT upregulation accompanied by Src activation (8, 17). We speculate that AKT is mainly controlled by another molecule that negatively interacts with Src, although further studies are needed. Constitutive pSTAT3 is also a common reason for trastuzumab resistance (27). Phosphorylation of STAT3 was enhanced in NCI-N87HR and SNU2670HR cell lines that harbored high levels of Src phosphorylation. Moreover, CDK4/6, Bcl-2, ribosomal S6, and altered S-nitrosothiol homeostasis have been considered as resistance mechanisms against trastuzumab (26, 28–35).

In this study, we confirmed that Src contributed to trastuzumab resistance, but we have not determined the reason why Src was activated in some HR cell lines and not in others. As the next step, we should uncover the mechanism of Src activation. According to an early study, Src mutation can cause Src activation in lapatinib (dual EGFR and HER2 tyrosine kinase inhibitor)-resistant cells (36). Based on our comprehensive understanding of Src, it is considered as a very rare event. The other major molecules that facilitate Src activation are integrins and transmembrane RTKs (14). Additionally, ligands such as substance P activate Src by increasing expression of neurokinin-1 receptor, thereby facilitating HER2 and EGFR dimerization (37). Several lines of evidence have suggested that resistance against HER2-targeting drugs is associated with epithelial-to-mesenchymal transition, and the resistant cells are more sensitive to Src inhibitors (35).

In our study, SNU216HR and SNU2773HR cell lines showed reduced expression levels of pSrc. However, pFAK was elevated in these cell lines (Fig. 1B). Bosutinib inhibited Src-dependent FAK tyr925 phosphorylation to mediate the downstream molecules. Furthermore, the level of E-cadherin was elevated in these cell lines (Supplementary Fig. S1). Therefore, in HR cell lines through activation of Src or FAK, the Src inhibitor acts to reverse the resistance.

In conclusion, Src contributes to trastuzumab resistance in HER2-positive gastric and biliary tract cancer cells, and an Src-targeting strategy could be a potential measure to overcome trastuzumab resistance.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: M.H. Jin, Y.-J. Bang

Development of methodology: M.H. Jin, Y.-J. Bang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M.H. Jin, Y.-J. Bang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.H. Jin, Y.-J. Bang

Writing, review, and/or revision of the manuscript: M.H. Jin, Y.-J. Bang, D.-Y. Oh

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.H. Jin, A.-R. Nam, J.E. Park, J.-H. Bang

Study supervision: D.-Y. Oh

Grant Support

This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number 2016R1D1A1A09918133) and a grant from SNU Invitation Program for Distinguished Scholar to Dr. D.-Y. Oh.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received October 11, 2016.
  • Revision received November 21, 2016.
  • Accepted February 4, 2017.

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Resistance Mechanism against Trastuzumab in HER2-Positive Cancer Cells and Its Negation by Src Inhibition
Mei Hua Jin, Ah-Rong Nam, Ji Eun Park, Ju-Hee Bang, Yung-Jue Bang and Do-Youn Oh
Mol Cancer Ther June 1 2017 (16) (6) 1145-1154; DOI: 10.1158/1535-7163.MCT-16-0669
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