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. 2015 Jul 15;16(10):1535–1547. doi: 10.1080/15384047.2015.1071737

The small GTPase ADP-Ribosylation Factor 1 mediates the sensitivity of triple negative breast cancer cells to EGFR tyrosine kinase inhibitors

Eric Haines 1, Sabrina Schlienger 1, Audrey Claing 1,*
PMCID: PMC4846185  PMID: 26176330

Abstract

The clinical use of EGFR-targeted therapy, in triple negative breast cancer patients, has been limited by the development of resistance to these drugs. Although activated signaling molecules contribute to this process, the molecular mechanisms remain relatively unknown. We have previously reported that the small GTPase ADP-Ribosylation Factor 1 (ARF1) is highly expressed in invasive breast cancer cells and acts as a molecular switch to activate EGF-mediated responses. In this study, we aimed at defining whether the high expression of ARF1 limits sensitivity of these tumor cells to EGFR inhibitors, such as gefitinib. Here, we show that the knock down of ARF1 expression or activity decreased the dose and latency time required by tyrosine kinase inhibitors to induce cell death. This may be explained by the observation that the depletion of ARF1 suppressed gefitinib-mediated activation of key mediators of survival such as ERK1/2, AKT and Src, while enhancing cascades leading to apoptosis such as the p38MAPK and JNK pathways, modifying the Bax/Bcl2 ratio and cytochrome c release. In addition, inhibiting ARF1 expression and activation also results in an increase in gefitinib-mediated EGFR internalization and degradation further limiting the ability of this receptor to promote its effects. Interestingly, we observed that gefitinib treatment resulted in the enhanced activation of ARF1 by promoting its recruitment to the receptor AXL, an important mediator of EGFR inhibition suggesting that ARF1 may promote its pro-survival effects by coupling to alternative mitogenic receptors in conditions where the EGFR is inhibited. Together our results uncover a new role for ARF1 in mediating the sensitivity to EGFR inhibition and thus suggest that limiting the activation of this GTPase could improve the therapeutic efficacy of EGFR inhibitors.

Keywords: ADP-ribosylation factor 1, breast cancer cells, epidermal growth factor receptor, resistance, tyrosine kinase inhibitors

Abbreviations

ARF1

ADP-Ribosylation Factor

EGF

Epidermal Growth Factor

EGFR

Epidermal Growth Factor Receptor

RTK

Receptor Tyrosine Kinase

TKi

Tyrosine Kinase inhibitor

TNBC

Triple negative breast cancer.

Introduction

The triple negative breast cancer (TNBC) subtype is characterized by the lack of expression of the estrogen, progesterone and HER2 receptors. Approximately 15-20% of global breast cancers are diagnosed as TNBC.1 This breast cancer subtype is considered to have an aggressive phenotype with high histological grade and metastatic potential.2,3 Moreover, disease recurrence has been shown to occur earlier in TNBC patients.4 This results in an overall poor patient prognosis.5 Since current cytotoxic chemotherapeutics have shown to be effective only in a small proportion of patients,6 there are present attempts to identify and characterize agents that therapeutically target specific oncogenic factors.

The epidermal growth factor receptor (EGFR) is highly expressed in the majority of TNBC patients,7 and associated with a poor prognosis making this receptor tyrosine kinase (RTK) a potential therapeutic target for the treatment of this aggressive form of breast cancer.7,8 Since the EGFR and HER2 are the 2 EGFR family members best characterized for their role in cancer, the majority of drugs targeting the EGFR family blocks these 2 members. There are 2 predominant types of EGFR-targeted therapies: monoclonal antibodies targeting the extracellular domain of the receptor and tyrosine kinase inhibitors targeting the kinase activity of the receptor.9,10 However, little to no therapeutic benefits have been observed in recent attempts at targeting the EGFR in TNBC patients.11,12 The development of EGFR inhibitor resistance (either innate or acquired) has been shown to play a major impact on the lack of response observed in these patients.13 Multiple mechanisms of resistance such as mutations in the EGFR itself and its downstream signaling effectors, increased expression of receptor tyrosine kinases (RTKs) (EGFR, HER2-3, AXL, cMET), and activation of other signaling regulators (Src, ERK1/2, AKT) have been described in the literature.12,14-19 Inhibiting these mechanisms of acquired resistance is an effective strategy to improve the sensitivity of these patients to EGFRTKis.20-22 In fact, the inhibition of the ERK1/2 pathway, as well as the AXL and cMET receptors have been shown to decrease cell growth and tumor formation of gefitinib-resistant cancer cells.20-22 However, the underlying mechanisms of acquired EGFRTKi resistance in highly invasive breast cancer cells have yet to be fully characterized.

Recently, we demonstrated that ADP-Ribosylation Factors 1 (ARF1), one of the 6 identified ARF isoform (ARF1 to 6) members of the superfamily of Ras GTPases, is activated downstream of the EGFR in MDA-MB-231 cells, a cellular model of TNBC.23 ARF proteins are broadly known for their role in vesicular trafficking, membrane lipid remodeling and reorganization of the actin cytoskeleton. Of the ARF isoforms, ARF1 and ARF6 are the best characterized. Like all GTPases, these proteins are inactive when bound to GDP, but become active when GTP is loaded by specific guanine nucleotide exchange factors (GEF). While ARF1 was first identified as a key regulator of Golgi trafficking, in the most invasive breast cancer cell lines, we reported that this ARF isoform was overexpressed and localized to the plasma membrane where it could be activated by the EGFR to control signaling to the PI3K survival pathway.23 We further demonstrated that activation of this protein following EGF stimulation is dependent upon the recruitment of the classical EGFR adaptor proteins, Grb2 and p66Shc.24 Depletion of ARF1 markedly impairs migration, invasion and proliferation of highly invasive breast cancer cells.23,25,26

In this study, we aimed at defining whether this small GTP-binding protein could also play a role in mediating the sensitivity of TNBC cells to EGFR tyrosine kinase inhibitors (EGFRTKis) since it is activated by the EGFR and acts to regulate numerous downstream signaling events coordinating key physiological responses characteristic of tumor cells and associated with invasiveness. Here, we report that ARF1 plays a key role in mediating EGFRTKi sensitivity. The knockdown or inhibition of this GTPase activity significantly improved the sensitivity of breast cancer cells to gefitinib. Our results suggest that targeting this key protein in combination with EGFR inhibitors may enhance their effectiveness and efficiency.

Results

ARF1 knockdown sensitizes breast cancer cells to gefitinib treatment

We have recently identified ARF1 as a key downstream effector of EGFR signals,23,24 and asked whether this small GTP-binding protein could mediate the sensitivity of breast cancer cells to EGFRTKis. We first used gefitinib-resistant TNBC cells that highly express the EGFR and ARF124 to examine the role of this small GTP-binding protein in mediating gefitinib sensitivity. In accordance with previously published findings,29-31 MDA-MB-231 cells were resistant to clinically relevant doses of geftinib (0.1, 1, 10 μM).27,28 However, a decrease in viability was observed in cells treated with high doses of this inhibitor (25, 50 μM) (Fig. 1A).27,29-31 Interestingly, the knockdown of ARF1 levels using 2 different siRNAs, significantly reduced viability at doses of gefitinib ranging from 1 to 50 μM. The IC50 for gefitinib treatment were 34.4 μM in control cells and 19.1 and 16.7 μM in ARF1 siRNA #1 and ARF1 siRNA #2 transfected cells, respectively (Table 1). A cell counting assay confirmed that the depletion of ARF1 was an effective strategy to sensitize cells to gefitinib (Fig. 1B). The depletion of ARF1 sensitized 2 other TNBC cell lines known to express high levels of both EGFR and ARF1, HCC70 and MDA-MB-157 cells, as well as the HER2-positive SKBR3 cell line to EGFR inhibition (Figs. S1A-C, Table 1). Whereas, the ER-positive, low EGFR/ARF1 expressing MCF7 cells were sensitive to EGFR inhibitors, but the knockdown of this ARF isoform had no effect on gefitinib sensitivity (Fig. S1D; Table 1).24 To demonstrate that the effects we observed were specific, we next performed a rescue experiment and overexpressed an ARF1 cDNA mutant (ARF1Mut) that contained the same sequence as the wild type ARF1, but was not targeted by the siRNA. As shown in Figure 1C and D, the enhanced sensitivity to gefitinib observed in ARF1-depleted cells was reversed upon the overexpression of ARF1Mut. Finally, the effect of gefitinib (10 μM) was further examined at varying times of exposure. As illustrated in Figure 1F, the knockdown of ARF1 potentiated the effect of this inhibitor in all examined time points (12 – 72 hours). All doses used in these experiments significantly inhibited EGF-dependent EGFR, ERK1/2 and AKT activation in all tested cell types (Fig. S2).

Figure 1.

Figure 1.

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ARF1 mediates gefitinib sensitivity in invasive breast cancer cells. (A) Percent cell death was assessed by a MTT assay in MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated 24 hours with indicated concentrations of gefitinib. Western blot analysis confirmed the depletion of ARF1. (B) Percent cell death was assessed by a cell counting assay in MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated 24 hours with indicated concentrations of gefitinib. (C) Percent cell death was assessed by a MTT assay in MDA-MB-231 cells that were transfected with CTL siRNA, ARF1 siRNA alone or ARF1 siRNA and ARF1Mut cDNA and then treated 24 hours with indicated concentrations of gefitinib. Western blot analysis confirmed the depletion of ARF1 and the expression of HA-tagged ARF1Mut. (D) Percent cell death was assessed by a cell counting assay in MDA-MB-231 cells that were transfected with CTL siRNA, ARF1 siRNA alone or ARF1 siRNA and ARF1Mut cDNA and then treated 24 hours with indicated concentrations of gefitinib. (E) Percent cell death was assessed by a MTT assay in MCF7 cells that were transfected with CTL or HA-tagged ARF1 cDNA and then treated 24 hours with indicated concentrations of gefitinib. Western blot analysis confirmed the expression of HA-tagged ARF1. (F) Percent cell death of MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for indicated time points as assessed by a MTT assay. (G) Percent cell death was assessed by a MTT assay in MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated 24 hours with indicated concentrations of lapatinib. (H) Percent cell death of SKBR3 cells as assessed as in (G). For all experiments, data shown are mean ±Standard error the mean (SEM). Significance was measured by a 2-way ANOVA with n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001.

Table 1.

Effect of ARF1 depletion on the IC50 of EGFRTKis in breast cancer cells. The IC50 for control cells or ARF1 knockdown cells treated with either gefitinib, tivantinib, R428 or lapatinib for 24 hours. Data shown are mean values. Significance was measured using an unpaired, 2-tailed T-test with n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001.

Cell Line Control Condition Experimental Condition Inhibitor IC50 Control (µM) IC50 Experimental (µM)
MDA-MB-231 CTL siRNA ARF1 siRNA #1 Gefitinib 34.37 19.06**
MDA-MB-231 CTL siRNA ARF1 siRNA #2 Gefitinib 34.37 16.72**
MDA-MB-231 CTL siRNA ARF1 siRNA #1 Lapatinib 9.74 2.46*
MDA-MB-231 CTL siRNA ARF1 siRNA #1 Tivantinib 40.48 38.37
MDA-MB-231 CTL siRNA ARF1 siRNA #1 R428 5.46 5.85
MDA-MB-231 DMSO Brefeldin A Gefitinib 45.96 18.53*
HCC70 CTL siRNA ARF1 siRNA #1 Gefitinib 53.14 12.95*
MDA-MB-157 CTL siRNA ARF1 siRNA #1 Gefitinib 46.46 14.04**
SKBR3 CTL siRNA ARF1 siRNA #1 Gefitinib 1.07 0.49**
SKBR3 CTL siRNA ARF1 siRNA #1 Lapatinib 23.63 8.39***
MCF7 CTL siRNA ARF1 siRNA #1 Gefitinib 18.56 20.74
MCF7 Vector HA-ARF1 cDNA Gefitinib 20.15 60.86***
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To further demonstrate the importance of ARF1 in mediating gefitinib sensitivity, we overexpressed an HA-Tagged ARF1 cDNA in the gefitinib-responsive MCF7 cell line. In these experiments, ARF1 overexpression reduced gefitinib sensitivity compared to control cells (Fig. 1E). The IC50 for gefitinib treatment were 20.2 μM in control cells and 60.9 μM in ARF1 overexpressing cells, respectively (Table 1). These results were confirmed using a cell counting assay (Fig. S1E). Together, these results suggest that ARF1 mediates the efficacy of gefitinib to kill tumor cells.

Because compounds blocking ARF1 activity as well as mutants mimicking the inactive and active forms of ARF1 have been useful in demonstrating the function this small GTPase plays in cells, we further examined sensitivity to RTK is using these alternative approaches. First, treatment of the MDA-MB-231 cells with Brefeldin A (BFA, 10 nM), an ARFGEF inhibitor,32 induced 50% cell death due to the induction of apoptosis, a well-documented effect of this compound.33 However, BFA treatment markedly synergized with gefitinib as indicated by the Chou-Talalay combination index equation (Figs. S3A-B).34,35 Additionally, similar effects were observed in cells overexpressing the inactive ARF1 mutant (ARF1TN) (Fig. S3C). Together, these findings demonstrate that the activity of ARF1 is essential in mediating gefitinib sensitivity of invasive breast cancer cells.

To better understand the role of ARF1 in mediating resistance to RTKi in general, we examined other EGFR and RTK inhibitors. Control and ARF1-depleted MDA-MB-231 cells were treated first with lapatinib, a dual EGFR/HER2 inhibitor, for 24 hours at doses ranging from 0.1 to 50 μM (Fig. 1G). While this drug induced approximately 50% cell death in control cells at a concentration of 10 μM, a similar effect was observed in ARF1-depleted conditions, but at a dose of 1 μM. The IC50 for lapatinib was therefore 9.7 μM in control conditions and 2.5 μM in ARF1 knockdown cells (Table 1). We observed a similar effect in the HER2-positive SKBR3 cell line (IC50: 23.6 μM for controls versus 8.4 μM for ARF1 depleted cells; Fig. 1H, Table 1). Finally, we noticed that ARF1-depletion had no effect on the sensitivity of MDA-MB-231 to both tivantinib and R428, cMet and AXL inhibitors, respectively (Fig. S1F, Table 1). Altogether, our results demonstrate that ARF1 plays a key role in mediating the sensitivity of TNBC and HER2-positive breast cancer cells to EGFRTKi, but not all RTK inhibitors.

ARF1 promotes gefitinib-mediated survival signals while blocking apoptosis

Next, we sought to define the molecular mechanisms by which ARF1 mediated resistance. It is generally accepted that activation of certain signaling mediators such as ERK1/2 and Src contribute to EGFRTKi sensitivity, although the exact mechanism remains unclear.16,19 We therefore next examined the activation of these signaling pathways in gefitinib-treated, ARF1-depleted cells. Drug treatment for up to 72 hours induced the activation of both ERK1/2 and Src in control cells (Fig. 2A). ARF1 depletion however delayed these signaling events. While gefitinib treatment of control cell was associated with a decreased AKT activation over time, this inhibition was more substantial and occurred earlier in cells depleted of ARF1 (24 hours vs. 48 hours in control cells) demonstrating that cell survival is mostly affected in gefitinib-treated-ARF1 knockdown conditions. Next, we evaluated the activation of these pathways in MCF7 cells overexpressing ARF1 upon gefitinib treatment (Fig. 2B). In these cells, gefitinib only slightly enhanced ERK1/2 activation while inhibiting AKT phosphorylation. Interestingly, overexpression of ARF1 markedly enhanced the effect of this EGFRTKi on the MAPK pathway while blocking the inactivation of AKT further supporting a role for ARF1 as a key mediator regulating survival signals upon gefitinib treatment.

Figure 2.

Figure 2.

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Gefitinib-induced survival signaling is altered in ARF1 depleted cells. (A) Western blot analysis utilizing phospho-specific antibodies was used to measure the activation of ERK1/2, AKT and Src in cell lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for the indicated time points. Data is presented as mean fold over basal activation ± SEM with n=3. Significance was measured by a 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. (B) Western blot analysis utilizing phospho-specific antibodies was used to measure the activation of ERK1/2 and AKT in cell lysates obtained from MCF7 cells that were transfected with CTL or HA-tagged ARF1 cDNA and then treated with 10 μM gefitinib for 24 hours. Data is presented as mean fold over basal activation ±SEM with n=3. Significance was measured by a 2-way ANOVA; *P < 0.05. (C) MDA-MB-231 percent cell death was assessed via a MTT assay in cells that were transfected with CTL or ARF1 siRNA and then treated with either PD0325901 (10 μM), LY294002 (15μM) or PP2 (1 μM) alone or in combination with gefitinib (10 μM) for 24 hours. Data shown are mean ± SEM. Significance was measured by a 2-way ANOVA with n = 3; *P < 0.05, ***P < 0.001.

The co-administration of specific inhibitors of the MAPK and PI3K/AKT pathways, in combination with EGFRTKis, was reported to be an effective strategy to improved clinical outcomes.36-38 Here, we therefore examined whether the depletion of ARF1 could further enhance the synergy between gefitinib and a MEK (PD0325901), a PI3Kinase (LY294002) and a Src kinase inhibitor (PP2). While all the inhibitors, when used alone, significantly reduced the viability of MDA-MB-231 cells, their effects were not altered by the depletion of ARF1 (Fig. 2C). Interestingly, the depletion of ARF1 significantly enhanced the effects of the co-treatment of gefitinib and the MEK inhibitor as well as the Src inhibitor, but not the PI3Kinase (Fig. 2C). We next confirmed these findings using the ARF inhibitor, BFA. Cotreatment with BFA significantly enhanced the induction of cell death induced by both LY294002 and PP2, but not PD0325901. More interestingly, a significant increase in cell death was observed in cells treated with the combination of BFA, gefitinib and PP2, but not LY294002 and PD0325901 compared to cells treated with only BFA and gefitinib (Figs. S3D, E, F). Together, our results suggest that targeting ARF1 can enhance the sensitivity to gefitinib alone, but it can also enhance the effect of co-treatment of this EGFRTKi with other clinically relevant inhibitors such as the Src kinase inhibitors.

Figure 3.

Figure 3.

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Enhanced gefitinib-mediated apoptotic signals in ARF1 depleted cells. (A) Western blot analysis utilizing phospho-specific antibodies was used to measure the activation of p38MAPK and pJNK in cell lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for the indicated time points. Data is presented as mean fold over basal activation ±SEM with n = 3. Significance was measured by a 2-way ANOVA; *P < 0.05, **P<0.01, ***P < 0.001. (B) Western blot analysis utilizing phospho-specific antibodies was used to measure the activation of p38MAPK and pJNK in cell lysates obtained from MCF7 cells that were transfected with CTL or HA-tagged ARF1 cDNA and then treated with 10 μM gefitinib for 72 hours. Data is presented as mean fold over basal activation ±SEM with n = 3. Significance was measured by a 2-way ANOVA; *P < 0.05, ***P < 0.001. (C) The expression of Bcl−2 and Bax was measured by western blot analysis in cell lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then left untreated or treated with 10 μM gefitinib for 72 hours. Data is presented as the mean ratio of Bax expression over Bcl-2 expression ± SEM with n=3. Significance was measured by a 2-way ANOVA; **P < 0.01. (D) The cytoplasmic and mitochondrial expression of Cytochrome C were measured by western blot analysis in cell lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then left untreated or treated with 10 μM gefitinib for 72 hours. Data is presented as mean fold Cytochrome C release ±SEM with n = 3. Significance was measured by a 2-way ANOVA; **P < 0.01.

With ARF1 promoting the activation of survival cascades in gefitinib treated MDA-MB-231 cells, we next examined the importance of this GTPase in the induction of gefitinib-mediated apoptotic signals through both p38MAPK and JNK.39,40 Therefore, we next investigated the role of ARF1 in mediating the activation of these pathways, upon gefitinib treatment. As shown in Figure 3A, control cells treated with gefitinib-induced the activation of both p38MAPK and JNK. Interestingly, the activation of these kinases was augmented in ARF1-depleted cells compared to control conditions suggesting that ARF1 may prevent gefitinib-dependent activation of these apoptotic pathways. Alternatively, we examined activation of these pathways in ARF1 overexpressing MCF7 cells. As illustrated in Figure 3B, ARF1 overexpression reduced gefitinib-induced activation of the apoptotic p38MAPK and JNK pathways compared to control conditions.

To confirm the role of ARF1 in regulating apoptotic pathways, we next examined the expression profile of specific markers. As shown in Figure 3C, we found that the Bax to Bcl2 protein expression ratio, an indicator of apoptosis, was significantly increased only in ARF1-depleted cells treated with gefitinib. Additionally, knockdown of ARF1 was associated with an increased release of mitochondrial Cytochrome C into the cytoplasm (Fig. 3D).

Altogether, these results suggest that high ARF1 expression, in highly invasive breast cancer cells, regulates anti-apoptotic pathways while promoting signals leading to cell survival.

ARF1 is essential for gefitinib-induced EGFR function

In our attempt to further understand the role ARF1 plays in EGFRTKi resistance, we focused on the function of the receptor itself. An increased expression and activation of the EGFR and other members of the EGFR family have been reported to limit EGFRTKi sensitivity.10,17,41 Here, we examined the expression of these receptors upon gefitinib treatment (10 μM) for up to 72 hours. In control MDA-MB-231 cells, an increase in EGFR expression was observed following 12 and 24 hours of treatment with the inhibitor, followed by a return to basal EGFR expression by 48 hours (Fig. 4A). Meanwhile, a reduction in HER2 levels was observed, whereas, HER3 levels remained stable. Levels of HER4 in MDA-MB-231 cells were undetectable. In ARF1-depleted conditions, gefitinib treatment no longer increased EGFR expression. Similarly, a decrease in HER2 expression at both 48 and 72 hours was also observed in cells depleted of ARF1 compared to control cells. No difference in HER3 expression was observed. These findings were next confirmed using the ARF inhibitor (Fig. S3G). We observed a significant decrease in EGFR and HER2 expression in cells co-treated with BFA and gefitinib. These results suggest that ARF1 may act to limit the sensitivity to gefitinib by decreasing the expression levels of both EGFR and HER2.

Figure 4.

Figure 4.

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Gefitinib-induced EGFR family member expression is mediated by ARF1 expression. (A) The protein expression of EGFR, HER2, HER3 and HER4 was assessed in lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for the indicated time points using western blot analysis. Data is presented as mean fold over basal ±SEM with n = 3. Significance was measured by a 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. (B) Western blot analysis was used to measure the expression of EGFR, HER2, HER3 and HER4 in cell lysates obtained from MCF7 cells that were transfected with CTL or HA-tagged ARF1 cDNA and then treated with 10 μM gefitinib for 72 hours. Data is presented as mean fold over basal expression ±SEM with n = 3. Significance was measured by a 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001.

Next, we examined the expression of EGFR family members in MCF7 cells. In control conditions, gefitinib treatment increased both EGFR and HER3 expression. However, a reduction in HER2 expression was observed upon gefitinib treatment (Fig. 4B). While a basal increase in EGFR and HER3 expression was detected in cells overexpressing ARF1, this ARF isoform was shown to only enhance gefitinib-mediated HER3 expression in this cell line. Moreover, the gefitinib-induced reduction in HER2 was reduced in ARF1-expressing MCF7 cells. No effect of ARF1 expression and gefitinib treatment on HER4 expression was observed. This further demonstrates the importance of ARF1 in mediating the expression of EGFR family members and that ARF1 may regulate gefitinib sensitivity in MCF7 cells by promoting signals downstream of HER2 and HER3.

Knowing that the depletion of ARF1 enhanced the gefitinib-dependent downregulation of the EGFR, we next examined whether treatment with this EGFRTKi enhanced receptor internalization. As shown in Figure 5A, gefitinib treatment promoted the internalization of the EGFR in control cells. Interestingly, this response occurred much faster (5 minutes vs. 30 minutes in control cells) in ARF1-depleted cells suggesting that ARF1 may mediate gefitinib sensitivity by controlling the membrane levels of the EGFR. Next, we investigated whether the internalized EGFR was targeted for degradation. To do this, we utilized the proteosomal inhibitor, MG132. As depicted in Figure 5B, the downregulation of EGFR expression in ARF1-depleted cells treated with gefitinib was partially recovered upon proteosomal inhibition. These results suggest that ARF1 may block the degradation of the EGFR in response to gefitinib treatment and thus reduce the sensitivity of these cells to EGFR inhibition.

Figure 5.

Figure 5.

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Gefitinib-dependent EGFR internalization and degradation is enhanced by ARF1 depletion. (A) The protein expression of EGFR was assessed in membrane extracts obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for the indicated time points using western blot analysis. Data is presented as mean fold over basal ±SEM with n = 3. Significance was measured by a 2-way ANOVA; ***P < 0.001. (B) The protein expression of EGFR was assessed in lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib alone, 1 μM MG132 alone or the combination of gefitinib (10 μM) and MG132 (1 μM) for 24 hours using western blot analysis. Data is presented as mean fold over basal ±SEM with n = 3. Significance was measured by a 2-way ANOVA; ***P < 0.001. (C) The threonine phosphorylation of the EGFR was assessed in lysates obtained from MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated with 10 μM gefitinib for 72 hours using a phospho-specific antibody. Data is presented as mean fold over basal ±SEM with n = 3. Significance was measured by a 2-way ANOVA; **P < 0.01. (D) MDA-MB-231 percent cell death was assessed via a MTT assay in cells that were transfected with CTL or ARF1 siRNA and then treated with SD220025 (100 nM) alone or in combination with gefitinib (10 μM) for 24 hours. Data shown are mean ±SEM. Significance was measured by a one-way ANOVA with n = 3; **P < 0.01.

Because ARF1-depletion enhanced gefitinib mediated p38MAPK activation (Fig. 3A) and this specific MAPK has been previously reported to promote the internalization of the EGFR through the threonine phosphorylation of residue T669 on the receptor,42 we next examine this molecular event. While gefitinib treatment enhanced the threonine phosphorylation of the EGFR in control cells, an increased phosphorylation was observed in ARF1-depleted conditions (Fig. 5C). These observations suggest that ARF1 may act to block the p38MAPK-dependent internalization of the EGFR and thus reduce the sensitivity of these cells to EGFR inhibition.

Finally, we determined whether the activation of p38MAPK was essential in mediating the cytotoxic properties of gefitinib in ARF1-depleted MDA-MB-231 cells. As shown in Figure 5D, we examined the induction of death in cells treated with the p38MAPK inhibitor, SB220025, alone or in combination with gefitinib. No difference in cell death was observed between ARF1-depleted and control cells treated with the p38MAPK inhibitor alone. As expected, treatment with gefitinib alone induced a 30% higher incidence of cell death in ARF1-depleted cells compared to control conditions. Remarkably, this gefitinib-dependent cell death was decreased upon the co-treatment with the p38MAPK inhibitor further emphasizing the importance of this ARF isoform in mediating signals through the p38MAPK pathway leading to EGFR internalization and the induction of apoptosis.

Together our results suggest that ARF1 mediates gefitinib sensitivity by blocking the internalization and degradation of the EGFR through a p38MAPK-dependent mechanism.

Gefitinib promotes the activation of ARF1 by enhancing its recruitment to the RTK AXL

Now that we have demonstrated that ARF1 plays an essential role in mediating gefitinib sensitivity, we asked whether gefitinib could in turn modulate the activity of this small GTPase. As shown in Figure 6A, treatment of MDA-MB-231 cells with this inhibitor resulted in an increased ARF1 activation. Similar effects were observed in HCC70 and MDA-MB-157 cells (Fig. 6 B, C).

Figure 6.

Figure 6.

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Gefitinib promotes ARF1 activation through the recruitment of this GTPase to AXL. (A) MDA-MB-231 cells were treated with indicated concentrations of gefitinib for 1 hour. A GST-GGA3 pulldown assay was used to capture activated ARF1 from cell lysates. Endogenous levels of activated ARF1 and the total protein levels of ARF1 were assessed by western blot analysis. Data shown are mean ±SEM. Significance was measured by a one-way ANOVA with n = 3; ***P < 0.001. (B) HCC70 cells were treated with indicated concentrations of gefitinib for 1 hour. ARF1 activation was assessed as described in (A). Data shown are mean ±SEM. Significance was measured by a one-way ANOVA with n=3; *P < 0.05, **P < 0.01. (C) MDA-MB-157 cells were treated with indicated concentrations of gefitinib for 1 hour. ARF1 activation was assessed as described in (A). Data shown are mean ±SEM. Significance was measured by a one-way ANOVA with n = 3; *P < 0.05, ***P < 0.001. (D) Co-immunoprecipitation experiments were used to assess the recruitment of ARF1 to the EGFR, HER2, cMet and AXL in MDA-MB-231 cells treated with gefitinib (10 μM) for 1 hour. Data is presented as mean receptor recruitment ±SEM with n = 3. Significance was measured by a 2-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. (E) MDA-MB-231 cells were treated with gefitinib (10 μM), lapatinib (10 μM), tivantinib (10 μM) and R428 (1 μM) alone or tivantinib (10 μM) and R428 (1 μM) in combination with gefitinib (10 μM) for 1 hour. ARF1 activation was assessed as described in (A). Data shown are mean ±SEM. Significance was measured by a one-way ANOVA with n = 3; **P < 0.01, ***P < 0.001. (F) Percent cell death was assessed by a MTT assay in MDA-MB-231 cells that were transfected with CTL or ARF1 siRNA and then treated 24 hours with gefitinib (10 μM), lapatinib (10 μM), tivantinib (10 μM) and R428 (1 μM) alone or tivantinib (10 μM) and R428 (1 μM) in combination with gefitinib (10 μM).

We next asked how gefitinib promoted the activation of ARF1 in MDA-MB-231 cells. As the expression and activity of other RTKs such as HER2, cMet and AXL have all been implicated in gefitinib resistance,13,18,29 we determined whether gefitinib treatment could enhance the recruitment, of ARF1 to these receptors. Indeed, gefitinib treatment was associated with an enhanced recruitment of ARF1 to HER2, cMet and AXL, but not to the EGFR (Fig. 6D). This would suggest that upon gefitinib treatment, other mitogenic receptors may promote ARF1 activation. Therefore, we first attempted to examine the necessity of these RTK in mediating gefitinib-induced ARF1 activation using pharmacological inhibitors. As shown in Figure 6E, the dual inhibition of EGFR and HER2 by lapatinib or the inhibition of cMET by tivantinib resulted in a similar degree of ARF1 activation compared to gefitinib treatment alone and neither blocked gefitinib-induced ARF1 activity. Interestingly, like in the EGFR inhibitor treatment, the AXL inhibitor, R428, was effective to enhance ARF1 activity. But, more importantly, this inhibitor blocked gefitinib-induced ARF1 activation. Finally, this observation was also found in MDA-MB-157 cells. As illustrated in Figure S4, gefitinib treatment resulted in increased ARF1 recruitment to AXL. Together, these findings suggest that in gefitinib treated invasive breast cancer cells, ARF1 is activated via its recruitment to AXL.

Finally, we determined whether ARF1 depletion could enhance the efficacy of co-inhibiting other RTKs with the EGFR. As previously shown, ARF1 depletion enhanced the efficacy of both gefitinib- and lapatinib-, but not tivantinib- and R428-treated MDA-MB-231 cells. Interestingly, a significant increase in cellular death was observed in ARF1-depleted cells co-treated with gefitinib and tivantinib compared to control conditions. However, in cells co-treated with the AXL inhibitor, R428, and gefitinib, the depletion of ARF1 was shown to have no effect further suggesting that ARF1 is signaling downstream of AXL in gefitinib treated cells (Fig. 6F).

These data therefore provide a mechanism by which activation of ARF1 may contribute to potentiate survival and signaling of mitogenic receptors in conditions where cells are treated with EGFRTKi. By continuously activating intermediates regulating EGFR expression, internalization and signaling ARF contributes to EGFRTKi resistance of highly invasive breast cancer cells.

Discussion

Although inhibitors of mitogenic receptor activity remain a therapy of choice to treat cancer, the development of resistance to these drugs, by numerous tumor cells, has greatly limited their broad use in patients. The research of strategies to overcome resistance has identified key events contributing together to this cellular response. However, the identification of the most upstream events and master regulators as well as the mechanisms whereby they mediate resistance has yet to be elucidated.

We have recently demonstrated that ARF1 is highly expressed in the most invasive types of breast cancer cells and that stimulation of the EGFR leads to activation of this molecular switch and ultimately proliferation, migration and invasion, by a mechanisms involving the recruitment of classical adaptor proteins.23-25 Here, we show a novel role for ARF1 in mediating EGFRTKi sensitivity of these tumor cells. The depletion or inhibition of this ARF isoform significantly enhanced the sensitivity of resistant invasive breast cancer cells to the EGFRTKi, gefitinib. In these conditions, clinically relevant doses of this inhibitor now become effective in inducing signals leading to cell death. We show that upon gefitinib treatment, ARF1 is activated upon its recruitment to the receptor AXL. This promotes the activation of survival signals through ERK1/2, Src and AKT, while blocking apoptotic signals through the p38MAPK and JNK pathways. Additionally, we demonstrate that ARF1 plays an important role in the internalization and degradation of the EGFR observed upon gefitinib treatment. Indeed, enhanced signals through the p38MAPK pathway enhanced the internalization and in turn, the degradation of EGFR in gefitinib-treated, ARF1-depleted cells (Fig. 7). From these data, we can conclude that ARF1 is an important regulator of EGFRTKi sensitivity in invasive breast cancer cells and its inhibition could improve therapeutic outcomes in patients treated with these drugs.

Figure 7.

Figure 7.

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The role of ARF1 in mediating gefitinib sensitivity in breast cancer cells. (A) Upon treatment with the EGFRTKi, gefitinib, ARF1 is recruited to the RTK, AXL, leading to the activation of this GTPase. Activated ARF1 promotes signals through the survival pathways, ERK1/2, Src and AKT. Furthermore, ARF1 attenuates gefitinib-induced apoptotic signals through p38MAPK pathway and JNK activation. Additionally, the actions of ARF1 on the p38MAPK mediate the internalization and degradation of the EGFR. Altogether, ARF1 mediates gefitinib sensitivity in breast cancer cells by promoting cell survival and EGFR stability.

Acquired resistance is a major factor that markedly reduces the efficacy of EGFRTKis in the clinical setting.5,14,43 A variety of mechanisms have been proposed to mediate this response of tumor cells. Firstly, modified expression and activation of the EGFR as well as mutations within the receptor have all been implicated in this process.44 It was reported that a point mutation (T790M), present within the kinase domain and targeted by EGFRTKis, significantly inhibited the functionality of these inhibitors. This mutation is commonly found in EGFRTKi treated patients.44 In this study, we have primarily used the MDA-MB-231 cell model, which does not possess this mutation.45 Our data indicate that treatment with gefitinib increased EGFR expression without affecting its activation. Interestingly, the depletion of ARF1 blocked this augmentation in EGFR expression and promoted its internalization and degradation.

It has also been proposed that EGFR family members can compensate for the loss of EGFR signals.44 In our cell model, EGFR, HER2 and HER3, but not HER4 are expressed. While EGFR expression was increased by gefitinib treatment, gefitinib decreased HER2 expression while not affecting HER3. However, a significant decrease in HER2 expression was observed in cells depleted of ARF1, and treated with gefitinib. Thus, ARF1 may block EGFRTKi sensitivity and promote resistance by stabilizing the expression both EGFR and HER2. It is important to note that other tyrosine kinase receptors have also been shown to be implicated in acquired resistance. For instance, the amplification of both the cMET and AXL receptors as well as their ligands hepatocyte growth factor (HGF) and Gas6, respectively, has been reported in EGFRTKi resistant cancers.18,44 In our experiments, no observable increase in cMET or AXL expression was detected in gefitinib-resistant MDA-MB-231 cells (Data not shown). We did however observe an increased activity of these 2 receptors, but this activation was shown to be independent of ARF1 expression. Additionally, we demonstrated that ARF1 is activated downstream of AXL in gefitinib-treated cells. Thus, ARF1 may mediate EGFR inhibitor sensitivity by propagating signals downstream of activated AXL. Others have also reported that an increased activation of the insulin growth factor receptor (IGFR) and the fibroblast growth factor receptor (FGFR) are associated with acquired resistance.44,46 Therefore, it would be of interest to examine the role of ARF1 downstream of these RTKs.

In addition to altered RTK signals, the activation of downstream key pathways have also been implicated in EGFRTKi resistance. In fact, point mutations in either Ras or PTEN, resulting in the constitutive activation of both the Ras/ERK1/2 and the PI3Kinase/AKT pathways, have been linked to drug resistance.47,48 Independent of these mutations, it has been shown that ERK1/2 can be reactivated via either a HER2- or Src-dependent mechanism; whereas, PI3Kinase/AKT activation has been primarily shown to be dependent of signals from either HER3 or MET receptor.19,44 In our gefitinib-insensitive MDA-MB-231 cells, we observed an increase ERK1/2 and Src activation and this activation was significantly reduced by ARF1 depletion. Furthermore, while gefitinib treatment was shown to reduce AKT phosphorylation in control cells, this decreased activation was found to be more pronounced in ARF1-depleted cells. This suggests that ARF1 may play an important role in drug sensitivity by activating survival pathways in TNBC cells.

Also, we observed an increase in p38MAPK and JNK activity in cells depleted of ARF1. The activation of these pathways have been linked to the gefitinib-induced cell death.49 Therefore, the increased gefitinib-sensitivity we observed in ARF1-depleted cells may stem from the enhance activation of these pathways. Furthermore, p38MAPK has been reported to promote the internalization of the EGFR, another mechanism known to promote EGFR inhibitor sensitivity of lung cancer cells.42,50 Indeed, for the first time in breast cancer cells, we observed an enhanced EGFR internalization in ARF1-depleted cells treated with gefitinib. Therefore, ARF1 may mediate gefitinib sensitivity by blocking p38MAPK signals to apoptosis and EGFR internalization

Although ARF1 plays key roles in physiology and diseases, successfully targeting small GTP-binding proteins as therapeutics targets remains a challenge. The design of molecular tools or drugs that specifically block the ability of a small G protein to become activated and interact with their effectors is of great interest.51,52 For ARF1, only a few inhibitors have been characterized in the literature.53. All of which have their limits regarding their potential use as therapeutics. Nevertheless, our demonstration that ARF1 is a key mediator of EGFRTKi sensitivity further support the relevance of studying the mechanisms by which ARF and other GTPases might control such phenomenon. Additionally, our demonstration that ARF1, namely, plays pleiotropic roles in tumorigenesis23,25,26 further supports the benefits of targeting this ARF isoform as an anti-cancer treatment. Here, we demonstrate that the small GTPase ARF1, a downstream molecular switch activated by the EGFR, is a key player in mediating the sensitivity of invasive breast cancer cells to the EGFRTKi, gefitinib. Future studies will focus on characterizing the importance of this GTPase in mediating EGFRTKi sensitivity in vivo, as well as examining the expression and activation profile of ARF1 in human tumor tissues isolated from patients that demonstrated resistance to EGFR inhibition. Our results thus far, suggest that while inhibiting ARF1 alone may have some therapeutic benefits such as reduced cancer cell proliferation, migration and invasion,23,25 a strategy where ARF1 would be inhibited together with EGFRTKis could serve to improve efficacy of a compound such as gefitinib by increasing its cellular sensitivity as well as possibly decreasing the incidence of acquired resistance.

Materials and Methods

Reagents and Antibodies

Lipofectamine 2000™ was purchased from Invitrogen (Burlington, Ontario, Canada). Epidermal growth factor was purchased from Fitzgerald Industries International, Inc.. (Concord, MA). Inhibitors used were gefitinib (Biovision Inc.. Milpitas, CA USA), tivantinib (Selleckchem, Houston, TX USA), R428 (Abmole Bioscience, Houston, TX), lapatinib, MG132, PD0325901, PP2, LY294002 and SB220025 (Sigma-Aldrich, Oakville, Ontario, Canada). Polyclonal antibodies used were EGFR, HER2, HER3, HER4, AXL, cMET, pAXL, pcMET, pErk1/2, pAKT, AKT, pSrc, pp38MAPK, p38MAPK, pJNK, JNK, pan-actin, Bax, Bcl2, Cytochrome C, CoxIV (Cell Signaling, Danvers, MA USA), ARF1 (Proteintech Group, Chicago, IL USA), HA-Tag, Erk1/2 (Santa Cruz Biotechnology, Dallas, TX USA). Monoclonal antibodies used were pan-PY (Santa Cruz Biotechnology), Src (Millipore, Etobicoke, Ontario, Canada). Other reagents used were goat anti-mouse antibody-horseradish peroxidase and goat anti-rabbit antibody-horseradish peroxidase (RD Systems, Minneapolis, MN USA) and Protein G-Agarose Plus beads (Santa Cruz Biotechnology).

DNA Plasmids and siRNAs

HA-tagged ARF1WT and ARF1WTMut cloned into a pcDNA3 vector, the double-stranded scrambled with 19-nucleotide duplex RNA and 2-nucleotide 3′ dTdT overhangs, ARF1 siRNA was previously described.23,25,54 All siRNAs include 2-nucleotide 3′ dTdT overhangs and were purchased from Dharmacon Inc. (Lafayette, CO USA).

Cell culture and Transfection

MDA-MB-231, MCF7, SKBR3, MDA-MB-157 cells were maintained at 37°C, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). HCC70 cells were maintained at 37°C, 5% CO2 in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% FBS. Cells were transfected with siRNA or plasmid DNA using Lipofectamine 2000™ according to the manufacturer's instructions. Briefly, cells were transfected with 25 nM siRNA for 72 hours prior to treatment with inhibitors at indicated concentrations and for indicated time points.

Co-immunoprecipitation and Western Blot Analysis

Cells from confluent 10 cm dishes were harvested in 700 μl of Lysis buffer (20 mM Tris-HCl pH 8, 1% Triton X-100, 10% glycerol, 140 mM NaCl, 5 mM EDTA, 1 nM sodium orthovanadate (Na3VO4) complemented with the protease inhibitors aprotinin (5 μg/ml), benzamidine (150 μg/ml), leupeptin (5 μg/ml), pepstatin (4 μg/ml) and phenylmethylsulfonyl fluoride (0.2 mg/ml). Lysates were solubilized at 4°C for 30 minutes and total soluble proteins were run on polyacrylamide gels and transferred onto nitrocellulose membranes. Proteins were than detected using indicated specific primary antibodies. Secondary antibodies were all horseradish peroxidase-conjugated, and chemiluminescence was used to visualize protein expression. The quantification of the digital images obtained was performed using ImageJ 1.46o software (National Institutes of Health, USA). For immunoprecipitation experiments, cell lysates described above were agitated with indicated antibodies and protein G-Agarose plus beads at 4°C for 3 hours. Proteins were eluted in SDS-sample buffer by heating to 65°C for 15 minutes. Protein interaction and tyrosine phosphorylation were measured by western blot analysis.

ARF Activation Assay

Cells were left untreated or treated with indicated concentrations of gefitinib for indicated time points. Activated ARF1 was measured as previously described.54 Briefly, cells were lysed in 400 μl of Lysis buffer E (pH 7.4, 50 mM Tris HCl, 1% NP-40, 137 mM NaCl, 10% glycerol, 5 mM MgCl2, 20 mM NaF, 1 mM NaPPi, 1 mM Na3VO4 and the protease inhibitors: aprotinin (5 μg/ml), benzamidine (150 μg/ml), leupeptin (5 μg/ml), pepstatin (4 μg/ml) and phenylmethylsulfonyl fluoride (0.2 mg/ml)). GST-GGA3-(1-316)55 coupled to glutathione-Sepharose 4B was added to each sample. The samples were then rotated at 4°C for 45 minutes. Proteins were eluted in 20 μl of SDS-sample buffer by heating to 65°C for 15 minutes. The detection of ARF1-GTP or ARF6-GTP was performed by western blot analysis using specific antibodies to ARF1 and ARF6, respectively.

Mitochondrial Fractionation

MDA-MB-231 cells were treated with indicated concentrations of gefitinib for 72 hours. Cells were collected and sonicated in CHM buffer (10 mM Tris-HCl pH 6.7, 10 mM KCl, 150 mM MgCl2). 0.25 M sucrose was added and cells were spun at 1000 g for 10 min and supernatant was collected as cytoplasmic fraction. Pellet was resuspended in SM buffer (10 mM Tris-HCl pH 6.7, 0.15 M MgCl2, 0.25 M sucrose) and spun 15 minutes at 5000 g. Mitochondrial pellet was lysed in MLB buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X 100, 0.3% NP-40). Cytoplasmic and mitochondrial Cytochrome C expression was assessed by western blot analysis.

Membrane Extraction

MDA-MB-231 cells were treated with indicated with gefitinib (10 μM) for indicated time points. Membrane extracts were isolated as previously described.56 Briefly, cells were harvested in an ice-cold hypotonic lysis buffer (10 mM Tris, pH 7.4, 1.5 mM MgCl2, 5 mM KCl, 1 mM dithiothreitol, 0.2 mM sodium orthovanadate, leupeptin (10 μg/ml), 4-(2-aminoethyl)benzenesulfonyl fluoride (400 μm), NaF (1 mM), pepstatin 1 μg/ml, aprotinin 1 mg/ml). Cells homogenates were centrifuged at 700 × g for 10 min to pellet nuclei and intact cells. Supernatants were spun at 100,000 × g for 30 min at 4°C to collect the membrane pellet. The pellet was lysed in hypotonic lysis buffer supplemented with 1% Nonidet P-40 before being spun at 100,000 × g for 30 min at 4°C. The expression of the EGFR in the supernatant was assessed by western blot analysis.

Cell Viability Assay

MTT assay was used as a measure of cell viability/death. Cells were transfected with CTL siRNA, ARF1 siRNA or ARF6 siRNA for 24 hours. Cells were then trypsinized and plated at confluency on a 96-well plate in medium supplemented with 10% FBS overnight. The next day, cells were left untreated or treated in serum-free medium with the specified concentrations of inhibitor for 12, 24, 48 or 72 hours, as indicated. Following the treatment, cells were stained with Thiazolyl Blue Tetrazolium Bromide (5 mg/ml) (Sigma-Aldrich) for 2 hours. The produced formazan product was than solubilized overnight in 20% SDS/50% Dimethyl-formamide solution (pH 4.7). Absorbance was measured at 570 nm with a reference wavelength at 450 nm using a plate reader. Cell counting assay used an equal cell number (1 × 104 cells) seeded in a 6-cm dish for 24h. For each indicated treatment, cells were trypsinized, stained with trypan blue, and live cells were manually counted.

Statistical Analysis

Statistical analysis was performed using either a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or a 2-way ANOVA followed by a Bonferroni's multiple comparison test using GraphPad Prism version 5 (San Diego, CA). The calculation of IC50 were also performed using GraphPad Prism version 5. CalcuSyn (Biosoft, Cambridge, Great Britain, UK) utilizing the Chou-Talalay combination index equation was used to calculate synergic relationships between tested inhibitors.

Funding

This work was supported by the Canadian Institutes of Health Research (grant number: MOP-106596 to AC). EH is the recipient of the Canadian Institutes of Health Research Fredrick Banting and Charles Best Doctoral Award. AC is the recipient of a Senior Scientist Award from the Fonds de Recherche du Québec- Santé.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website

SUPPLEMENTARY DATA

References

  • 1.Bauer KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer 2007; 109:1721-8; PMID:17387718; http://dx.doi.org/ 10.1002/cncr.22618 [DOI] [PubMed] [Google Scholar]
  • 2.Fulford LG, Reis-Filho JS, Ryder K, Jones C, Gillett CE, Hanby A, Easton D, Lakhani SR. Basal-like grade III invasive ductal carcinoma of the breast: patterns of metastasis and long-term survival. Breast Cancer Res 2007; 9:R4; PMID:17217540; http://dx.doi.org/ 10.1186/bcr1636 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, et al.. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 2004; 10:5367-74; PMID:15328174; http://dx.doi.org/ 10.1158/1078-0432.CCR-04-0220 [DOI] [PubMed] [Google Scholar]
  • 4.Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 2007; 13:4429-34; PMID:17671126; http://dx.doi.org/ 10.1158/1078-0432.CCR-06-3045 [DOI] [PubMed] [Google Scholar]
  • 5.Andre F, Zielinski CC. Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents. Ann Oncol 2012; 23 Suppl 6:vi46-51; PMID:23012302; http://dx.doi.org/ 10.1093/annonc/mds195 [DOI] [PubMed] [Google Scholar]
  • 6.Liedtke C, Mazouni C, Hess KR, Andre F, Tordai A, Mejia JA, Symmans WF, Gonzalez-Angulo AM, Hennessy B, Green M, et al.. Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer. J Clin Oncol 2008; 26:1275-81; PMID:18250347; http://dx.doi.org/ 10.1200/JCO.2007.14.4147 [DOI] [PubMed] [Google Scholar]
  • 7.Corkery B, Crown J, Clynes M, O'Donovan N. Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer. Ann Oncol 2009; 20:862-7; PMID:19150933; http://dx.doi.org/ 10.1093/annonc/mdn710 [DOI] [PubMed] [Google Scholar]
  • 8.Rakha EA, Elsheikh SE, Aleskandarany MA, Habashi HO, Green AR, Powe DG, El-Sayed ME, Benhasouna A, Brunet JS, Akslen LA, et al.. Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin Cancer Res 2009; 15:2302-10; PMID:19318481; http://dx.doi.org/ 10.1158/1078-0432.CCR-08-2132 [DOI] [PubMed] [Google Scholar]
  • 9.Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 2006; 12:7242-51; PMID:17189395; http://dx.doi.org/ 10.1158/1078-0432.CCR-06-0646 [DOI] [PubMed] [Google Scholar]
  • 10.Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005; 5:341-54; PMID:15864276; http://dx.doi.org/ 10.1038/nrc1609 [DOI] [PubMed] [Google Scholar]
  • 11.Dickler MN, Cobleigh MA, Miller KD, Klein PM, Winer EP. Efficacy and safety of erlotinib in patients with locally advanced or metastatic breast cancer. Breast Cancer Res Treat 2009; 115:115-21; PMID:18496750; http://dx.doi.org/ 10.1007/s10549-008-0055-9 [DOI] [PubMed] [Google Scholar]
  • 12.Baselga J, Albanell J, Ruiz A, Lluch A, Gascon P, Guillem V, Gonzalez S, Sauleda S, Marimon I, Tabernero JM, et al.. Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol 2005; 23:5323-33; PMID:15939921; http://dx.doi.org/ 10.1200/JCO.2005.08.326 [DOI] [PubMed] [Google Scholar]
  • 13.Sohn J, Liu S, Parinyanitikul N, Lee J, Hortobagyi GN, Mills GB, Ueno NT, Gonzalez-Angulo AM. cMET Activation and EGFR-Directed Therapy Resistance in Triple-Negative Breast Cancer. J Cancer 2014; 5:745-53; PMID:25368674; http://dx.doi.org/ 10.7150/jca.9696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med 2008; 358:1160-74; PMID:18337605; http://dx.doi.org/ 10.1056/NEJMra0707704 [DOI] [PubMed] [Google Scholar]
  • 15.Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al.. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350:2129-39; PMID:15118073; http://dx.doi.org/ 10.1056/NEJMoa040938 [DOI] [PubMed] [Google Scholar]
  • 16.Mueller KL, Hunter LA, Ethier SP, Boerner JL. Met and c-Src cooperate to compensate for loss of epidermal growth factor receptor kinase activity in breast cancer cells. Cancer Res 2008; 68:3314-22; PMID:18451158; http://dx.doi.org/ 10.1158/0008-5472.CAN-08-0132 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Bhargava R, Gerald WL, Li AR, Pan Q, Lal P, Ladanyi M, Chen B. EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations. Mod Pathol 2005; 18:1027-33; PMID:15920544; http://dx.doi.org/ 10.1038/modpathol.3800438 [DOI] [PubMed] [Google Scholar]
  • 18.Meyer AS, Miller MA, Gertler FB, Lauffenburger DA. The receptor AXL diversifies EGFR signaling and limits the response to EGFR-targeted inhibitors in triple-negative breast cancer cells. Sci Signal 2013; 6:ra66; PMID:23921085 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ochi N, Takigawa N, Harada D, Yasugi M, Ichihara E, Hotta K, Tabata M, Tanimoto M, Kiura K. Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer. Exp Cell Res 2014; 322:168-77; PMID:24440771; http://dx.doi.org/ 10.1016/j.yexcr.2014.01.007 [DOI] [PubMed] [Google Scholar]
  • 20.Huang MH, Lee JH, Chang YJ, Tsai HH, Lin YL, Lin AM, Yang JC. MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib. Mol Oncol 2013; 7:112-20; PMID:23102728; http://dx.doi.org/ 10.1016/j.molonc.2012.09.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rho JK, Choi YJ, Kim SY, Kim TW, Choi EK, Yoon SJ, Park BM, Park E, Bae JH, Choi CM, et al.. MET and AXL inhibitor NPS-1034 exerts efficacy against lung cancer cells resistant to EGFR kinase inhibitors because of MET or AXL activation. Cancer Res 2014; 74:253-62; PMID:24165158; http://dx.doi.org/ 10.1158/0008-5472.CAN-13-1103 [DOI] [PubMed] [Google Scholar]
  • 22.Yoshida T, Okamoto I, Okamoto W, Hatashita E, Yamada Y, Kuwata K, Nishio K, Fukuoka M, Janne PA, Nakagawa K. Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification. Cancer Sci 2010; 101:167-72; PMID:19804422; http://dx.doi.org/ 10.1111/j.1349-7006.2009.01368.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Boulay PL, Cotton M, Melancon P, Claing A. ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells. J Biol Chem 2008; 283:36425-34; PMID:18990689; http://dx.doi.org/ 10.1074/jbc.M803603200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Haines E, Saucier C, Claing A. The adaptor proteins p66Shc and Grb2 regulate the activation of the GTPases ARF1 and ARF6 in invasive breast cancer cells. J Biol Chem 2014; 289:5687-703; PMID:24407288; http://dx.doi.org/ 10.1074/jbc.M113.516047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Schlienger S, Campbell S, Claing A. ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion. Mol Biol Cell 2014; 25:17-29; PMID:24196838; http://dx.doi.org/ 10.1091/mbc.E13-06-0335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Sabe H, Hashimoto S, Morishige M, Ogawa E, Hashimoto A, Nam JM, Miura K, Yano H, Onodera Y. The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis. Traffic 2009; 10:982-93; 3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li ZX, Qu LY, Wen H, Zhong HS, Xu K, Qiu XS, Wang EH. Mig-6 overcomes gefitinib resistance by inhibiting EGFR/ERK pathway in non-small cell lung cancer cell lines. Int J Clin Exp Pathol 2014; 7:7304-11; PMID:25400829 [PMC free article] [PubMed] [Google Scholar]
  • 28.McGovern UB, Francis RE, Peck B, Guest SK, Wang J, Myatt SS, Krol J, Kwok JM, Polychronis A, Coombes RC, et al.. Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Mol Cancer Ther 2009; 8:582-91; PMID:19276163; http://dx.doi.org/ 10.1158/1535-7163.MCT-08-0805 [DOI] [PubMed] [Google Scholar]
  • 29.Erjala K, Sundvall M, Junttila TT, Zhang N, Savisalo M, Mali P, Kulmala J, Pulkkinen J, Grenman R, Elenius K. Signaling via ErbB2 and ErbB3 associates with resistance and epidermal growth factor receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells. Clin Cancer Res 2006; 12:4103-11; PMID:16818711; http://dx.doi.org/ 10.1158/1078-0432.CCR-05-2404 [DOI] [PubMed] [Google Scholar]
  • 30.Ono M, Hirata A, Kometani T, Miyagawa M, Ueda S, Kinoshita H, Fujii T, Kuwano M. Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol Cancer Ther 2004; 3:465-72; PMID:15078990 [PubMed] [Google Scholar]
  • 31.Krol J, Francis RE, Albergaria A, Sunters A, Polychronis A, Coombes RC, Lam EW. The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells. Mol Cancer Ther 2007; 6:3169-79; PMID:18089711; http://dx.doi.org/ 10.1158/1535-7163.MCT-07-0507 [DOI] [PubMed] [Google Scholar]
  • 32.Niu TK, Pfeifer AC, Lippincott-Schwartz J, Jackson CL. Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi. Mol Biol Cell 2005; 16:1213-22; PMID:15616190; http://dx.doi.org/ 10.1091/mbc.E04-07-0599 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tseng CN, Hong YR, Chang HW, Yu TJ, Hung TW, Hou MF, Yuan SS, Cho CL, Liu CT, Chiu CC, et al.. Brefeldin A Reduces Anchorage-Independent Survival, Cancer Stem Cell Potential and Migration of MDA-MB-231 Human Breast Cancer Cells. Molecules 2014; 19:17464-77; PMID:25356567; http://dx.doi.org/ 10.3390/molecules191117464 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chou TC, Talalay P. Generalized equations for the analysis of inhibitions of Michaelis-Menten and higher-order kinetic systems with two or more mutually exclusive and nonexclusive inhibitors. Eur J Biochem 1981; 115:207-16; PMID:7227366; http://dx.doi.org/ 10.1111/j.1432-1033.1981.tb06218.x [DOI] [PubMed] [Google Scholar]
  • 35.Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22:27-55; PMID:6382953; http://dx.doi.org/ 10.1016/0065-2571(84)90007-4 [DOI] [PubMed] [Google Scholar]
  • 36.Normanno N, De Luca A, Maiello MR, Campiglio M, Napolitano M, Mancino M, Carotenuto A, Viglietto G, Menard S. The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J Cell Physiol 2006; 207:420-7; PMID:16419029; http://dx.doi.org/ 10.1002/jcp.20588 [DOI] [PubMed] [Google Scholar]
  • 37.Jokinen E, Laurila N, Koivunen JP. Alternative dosing of dual PI3K and MEK inhibition in cancer therapy. BMC Cancer 2012; 12:612; PMID:23259591; http://dx.doi.org/ 10.1186/1471-2407-12-612 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Montero JC, Seoane S, Ocana A, Pandiella A. Inhibition of SRC family kinases and receptor tyrosine kinases by dasatinib: possible combinations in solid tumors. Clin Cancer Res 2011; 17:5546-52; PMID:21670084; http://dx.doi.org/ 10.1158/1078-0432.CCR-10-2616 [DOI] [PubMed] [Google Scholar]
  • 39.Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, Ambrosino C, Benito M, Nebreda AR. P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol Biol Cell 2004; 15:922-33; PMID:14617800; http://dx.doi.org/ 10.1091/mbc.E03-08-0592 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Dhanasekaran DN, Reddy EP. JNK signaling in apoptosis. Oncogene 2008; 27:6245-51; PMID:18931691; http://dx.doi.org/ 10.1038/onc.2008.301 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Frolov A, Schuller K, Tzeng CW, Cannon EE, Ku BC, Howard JH, Vickers SM, Heslin MJ, Buchsbaum DJ, Arnoletti JP. ErbB3 expression and dimerization with EGFR influence pancreatic cancer cell sensitivity to erlotinib. Cancer Biol Ther 2007; 6:548-54; PMID:17457047; http://dx.doi.org/ 10.4161/cbt.6.4.3849 [DOI] [PubMed] [Google Scholar]
  • 42.Lambert S, Frankart A, Poumay Y. p38 MAPK-regulated EGFR internalization takes place in keratinocyte monolayer during stress conditions. Arch Dermatol Res 2010; 302:229-33; PMID:20039046; http://dx.doi.org/ 10.1007/s00403-009-1020-0 [DOI] [PubMed] [Google Scholar]
  • 43.Hosford SR, Miller TW. Clinical potential of novel therapeutic targets in breast cancer: CDK4/6, Src, JAK/STAT, PARP, HDAC, and PI3K/AKT/mTOR pathways. Pharmgenomics Pers Med 2014; 7:203-15; PMID:25206307 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wheeler DL, Dunn EF, Harari PM. Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nat Rev Clin Oncol 2010; 7:493-507; PMID:20551942; http://dx.doi.org/ 10.1038/nrclinonc.2010.97 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Gendreau SB, Ventura R, Keast P, Laird AD, Yakes FM, Zhang W, Bentzien F, Cancilla B, Lutman J, Chu F, et al.. Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647. Clin Cancer Res 2007; 13:3713-23; PMID:17575237; http://dx.doi.org/ 10.1158/1078-0432.CCR-06-2590 [DOI] [PubMed] [Google Scholar]
  • 46.Terai H, Soejima K, Yasuda H, Nakayama S, Hamamoto J, Arai D, Ishioka K, Ohgino K, Ikemura S, Sato T, et al.. Activation of the FGF2-FGFR1 autocrine pathway: a novel mechanism of acquired resistance to gefitinib in NSCLC. Mol Cancer Res 2013; 11:759-67; PMID:23536707; http://dx.doi.org/ 10.1158/1541-7786.MCR-12-0652 [DOI] [PubMed] [Google Scholar]
  • 47.Raponi M, Winkler H, Dracopoli NC. KRAS mutations predict response to EGFR inhibitors. Curr Opin Pharmacol 2008; 8:413-8; PMID:18619559; http://dx.doi.org/ 10.1016/j.coph.2008.06.006 [DOI] [PubMed] [Google Scholar]
  • 48.Sos ML, Koker M, Weir BA, Heynck S, Rabinovsky R, Zander T, Seeger JM, Weiss J, Fischer F, Frommolt P, et al.. PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res 2009; 69:3256-61; PMID:19351834; http://dx.doi.org/ 10.1158/0008-5472.CAN-08-4055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Lee JY, Lee YM, Chang GC, Yu SL, Hsieh WY, Chen JJ, Chen HW, Yang PC. Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy. PLoS One 2011; 6:e23756; PMID:21858220; http://dx.doi.org/ 10.1371/journal.pone.0023756 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Nishimura Y, Bereczky B, Ono M. The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines. Histochem Cell Biol 2007; 127:541-53; PMID:17361439; http://dx.doi.org/ 10.1007/s00418-007-0281-y [DOI] [PubMed] [Google Scholar]
  • 51.Spiegel J, Cromm PM, Zimmermann G, Grossmann TN, Waldmann H. Small-molecule modulation of Ras signaling. Nat Chem Biol 2014; 10:613-22; PMID:24929527; http://dx.doi.org/ 10.1038/nchembio.1560 [DOI] [PubMed] [Google Scholar]
  • 52.Vigil D, Cherfils J, Rossman KL, Der CJ. Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat Rev Cancer 2010; 10:842-57; PMID:21102635; http://dx.doi.org/ 10.1038/nrc2960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Bourgoin SG, El Azreq MA. Small inhibitors of ADP-ribosylation factor activation and function in mammalian cells. World J Pharmacol 2012; 1:55-64; http://dx.doi.org/ 10.5497/wjp.v1.i4.55 [DOI] [Google Scholar]
  • 54.Cotton M, Boulay PL, Houndolo T, Vitale N, Pitcher JA, Claing A. Endogenous ARF6 interacts with Rac1 upon angiotensin II stimulation to regulate membrane ruffling and cell migration. Mol Biol Cell 2007; 18:501-11; PMID:17122362; http://dx.doi.org/ 10.1091/mbc.E06-06-0567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Giguere P, Rochdi MD, Laroche G, Dupre E, Whorton MR, Sunahara RK, Claing A, Dupuis G, Parent JL. ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6. Cell Signal 2006; 18:1988-94; PMID:16650966; http://dx.doi.org/ 10.1016/j.cellsig.2006.03.003 [DOI] [PubMed] [Google Scholar]
  • 56.Deming PB, Campbell SL, Baldor LC, Howe AK. Protein kinase A regulates 3-phosphatidylinositide dynamics during platelet-derived growth factor-induced membrane ruffling and chemotaxis. J Biol Chem 2008; 283:35199-211; PMID:18936099; http://dx.doi.org/ 10.1074/jbc.M804448200 [DOI] [PMC free article] [PubMed] [Google Scholar]

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