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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Mol Cancer Ther. 2010 Apr 20;9(5):1432–1442. doi: 10.1158/1535-7163.MCT-10-0160

X-Linked Inhibitor of Apoptosis Protein Inhibits Apoptosis in Inflammatory Breast Cancer Cells with Acquired Resistance to an ErbB1/2 Tyrosine Kinase Inhibitor

Katherine M Aird 1,3, Rami B Ghanayem 2, Sharon Peplinski 2, Herbert K Lyerly 1,2,3, Gayathri R Devi 1,2,3
PMCID: PMC2957814  NIHMSID: NIHMS185921  PMID: 20406946

Abstract

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analog, ErbB1/2 inhibitor)-resistant IBC cells, we identified overexpression of an anti-apoptotic protein, XIAP, in acquired resistance to GW583340 in both ErbB2 overexpressing SUM190 and ErbB1 activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small molecule inhibitor (embelin), which abrogates the XIAP/procaspase 9 interaction, resulted in decreased viability, demonstrating that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents.

Keywords: IRES, embelin, survivin, FOXO3a, p-AKT

Introduction

Apoptotic dysregulation is a fundamental characteristic of cancer that allows transformed cells to survive and proliferate (1, 2). In part, this is due to defects in caspase activity, the execution phase of apoptosis. The inhibitor of apoptosis proteins (IAPs) are one of the major protein families that regulate caspase activation and programmed cell death (3). The family currently consists of eight members that are characterized by the presence of one or more baculoviral IAP repeat (BIR) domains and are highly conserved among mammalian and non-mammalian species (4).

In particular, one of the IAP proteins, X-linked inhibitor of apoptosis protein (XIAP), has been identified as the most potent caspase inhibitor to date (4). XIAP can bind and inhibit activation of procaspases 9, 7, and 3 (5). This leads to inhibition of both intrinsic (mitochondrial) and extrinsic (death receptor-mediated) pathways of apoptosis (3), which is not evident with another prominent anti-apoptotic protein Bcl-2, which inhibits cytochrome c release from the mitochondria but does not directly bind to caspases (6). In addition, XIAP mRNA has an internal ribosomal entry sequence (IRES) (7), which has been identified to be upregulated during cellular stress (8–10). XIAP is expressed in almost all tissues and cell types (11); however, it is often overexpressed in tumors versus normal tissue (12), including breast cancer (13), and has been strongly linked to therapeutic resistance in cervical, ovarian, and prostate cancers (14, 15). In addition to its caspase-binding function, XIAP has been observed to regulate the activity of key survival factors like AKT, nuclear factor kappa B (NF-κB), and another IAP family member, survivin (16). Therefore, there is a growing interest in targeting XIAP, and inhibitors of XIAP are currently being developed to help overcome resistance to mainstay therapies (17).

Recently, we reported a novel functional link between the epidermal growth factor receptor 2 (ErbB2) signaling pathway and XIAP in SUM190 cells, an ErbB2 overexpressing inflammatory breast cancer (IBC) cell line resistant to trastuzumab (an ErbB2 targeting monoclonal antibody) (18). IBC is an aggressive, fast-growing, and highly invasive cancer that is clinicopathologically distinct from a neglected locally advanced breast cancer (LABC) (19). IBC tumors are often resistant to chemo- and radio- therapy and therefore disease-free survival is poor (20, 21). ErbB2 is commonly overexpressed in IBC tumors; (22) however, the development of acquired resistance to FDA approved agents, trastuzumab (humanized ErbB2 monoclonal antibody) and lapatinib (a dual ErbB1/2 tyrosine kinase inhibitor), limits the clinical efficacy of these anti-ErbB2 therapeutic strategies (23–25). Clinical trials using lapatinib as a monotherapy have shown that it is effective in patients with ErbB2 overexpressing breast cancer that have been heavily pre-treated with other therapeutics including trastuzumab (26, 27) with response rates ranging from 7–35% (28). Interestingly, in IBC patients lapatinib has a greater efficacy with response rates ranging from 50–100% (29, 30). However, clinical studies with lapatinib as a monotherapy also indicate that clinical responses are generally short-lived in breast cancer patients (31), and acquired resistance is common. Previously reported mechanisms of lapatinib resistance include activation of estrogen receptor (ER) signaling (25), upregulation of the anti-apoptotic protein MCL-1 (32), and potentially the modulation of cancer cell metabolism (33). In the present study, we evaluated XIAP action in a model of acquired resistance to a lapatinib analog (GW583340) in both ErbB2 overexpressing and ErbB1 activated IBC cell lines wherein cells were chronically exposed to GW583340, similar to patients receiving daily doses of lapatinib when given as a monotherapy. Continuous exposure to GW583340 for more than 3 months converted the parental GW583340-sensitive IBC cells to being resistant to the apoptotic-inducing and growth inhibitory effects of the inhibitor. We identified XIAP overexpression to be the key difference between the parental GW583340-sensitive and GW583340-resistant IBC cell lines studied here. This overexpression was demonstrated to be mediated via translation of XIAP using its IRES in its 5’ untranslated region (UTR). Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. Further, XIAP downregulation using embelin (a small molecule inhibitor that interrupts the interaction between XIAP and procaspase 9) (34) caused reversal of GW583340-resistance in the acquired resistant IBC cellular models. The present study supports the observation that XIAP is required for the survival of IBC cells with acquired GW583340 resistance.

Materials and Methods

Cell culture

SUM149 and SUM190 cells were obtained from Asterand, Inc. (Detroit, MI). All cell lines were cultured as described previously (18). Laboratory grade lapatinib analog (GW583340; Sigma) was dissolved in DMSO. Acquired resistance to GW583340 was selected in SUM190 and SUM149 cells (referred to as rSUM190 and rSUM149) by culturing cells in normal growth media supplemented with increasing concentrations of GW583340 (0.25–2.5 µM and 0.25–7.5 µM, respectively). Initially, marked cell death and decrease in cell growth was observed in the cells. However, after two weeks of each increase in drug concentration, small colonies of viable cells were observed, which were cultured until confluence before the next increase in drug concentration. This was continued for a minimum of 3 months. From then on, both rSUM190 and rSUM149 cells were routinely cultured in 2.5 µM and 7.5 µM GW583340, respectively.

Generation of stable XIAP overexpressing IBC cell lines

SUM149 cells stably expressing wtXIAP and FG9 GFP vector control were generated using a lentiviral expression system (kindly provided by Dr. Colin Duckett, University of Michigan). Briefly, HEK294T cells were transfected using polyethylenimine with 5 µg of pHCMV, pRRE, and pRSVrev (35), which drive the expression of lentiviral structural proteins, and 5 µg of pFG9 EF1 XIAP WT silent mutation hyg or pFG9 EF1a hygro GFP (as a control) (36). Twenty-four hours post-transfection, media was changed. Forty hours post-transfection, the virus-containing media on the HEK293T cells was collected and filtered through a 0.45 mm Millex HV PVDF filter unit (Millipore) onto cells [with 25 mM polybrene (Sigma)] After four hours, fresh media was added and cells were incubated for an additional forty-eight hours at 37°C, 5% CO2. Stable cells were selected by the addition of hygromycin B (Invitrogen; 200 mg/mL).

Treatment of cells with agents for determination of cell counts and signaling

Cells were seeded in 6-well plates (Corning Incorporated, Corning, NY) and allowed to reach 70% confluence. Cells were treated for 24 h to 7 d in regular growth media with GW583340 (Sigma) or 48 h with embelin (Sigma, dissolved in DMSO) or PI3K inhibitor, LY294002 (40 µM, Sigma, dissolved in DMSO). DMSO (at the same concentration as drug treatments) was used as a vehicle control in all experiments. Cell counts were determined by trypan blue exclusion.

Determination of cell death

Cells were treated as described above and then stained for Annexin V and PI using the Annexin V Biotin Kit (Beckman Coulter, Fullerton, CA) as per the manufacturer’s instructions. Total cell death is presented as the sum of Annexin V+/PI−, Annexin V+/PI+, and Annexin V−/PI+ cells. Alternatively, for some experiments cell death was ascertained by staining with 7-AAD (Invitrogen, Carlsbad, CA) for 30 min. At least twenty-five thousand events were collected on a FACScalibur flow cytometer (Beckton Dickinson) and analyzed using Cellquest software (Beckton Dickinson).

Western Immunoblot Analysis

Western immunoblot analysis was carried out as described previously (18). Cells were harvested for western immunoblot analysis at 24 h (GW583340) or 48 h (embelin, LY294002) post-treatment. Membranes were incubated with primary antibodies against XIAP (BD Bioscience, San Jose, CA), procaspase 9 (NeoMarkers, Fremont, CA), actin, GAPDH, FOXO3a, Bcl-2 (Santa Cruz), survivin (R&D Systems, Minneapolis, MN), p-AKT (Ser473), AKT, p-ErbB2 (Tyr877), ErbB2, p-MAPK (Thr202/Tyr204), MAPK, ErbB1 (Cell Signaling), and total phosphotyrosine clone 4G10 (Upstate, Lake Placid, NY) overnight at 4°C. Detection of total protein was carried out in all experiments by stripping the same membrane as previously described (15). Densitometric analysis was performed using the NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Immunofluorescent microscopy

Cells were seeded onto cover slips (VWR, West Chester, PA) in dishes and allowed to reach 70% confluence. Cover slips were washed once with PBS and fixed with 100% methanol at −20°C for 20 min. After fixation, cells were blocked with 1% BSA/PBS at 37°C for 30 min and incubated with XIAP antibody (BD Biosciences) for 1 h at room temperature. Cells were then washed three times with PBS for 5 min and incubated with R-PE-labeled secondary antibody (Southern Biotech, Birmingham, AL) for 1 h at room temperature. For a nuclear counterstain, cells were incubated for 1 min with 0.1 µg/ml Hoechst 33258 (Sigma). Finally, cells were washed three times with PBS, cover slips were inverted onto slides, sealed, and imaged on a Zeiss Axio Observer inverted widefield fluorescence microscope using a 63x/1.40 DIC Plan Apochromat objective. Images were captured on a Hamamatsu ORCA ER CCD camera (Hamamatsu Corporation, Bridgewater, NJ). The system was controlled by MetaMorph Software (Molecular Devices, Downingtown, PA). XIAP staining intensity was measured by using NIH ImageJ software.

Real time polymerase chain reaction

Quantitative real-time PCR was performed as described previously (18). β-actin was used as an internal control. ΔΔCT shows the difference between actin control and XIAP. Folds (2−ΔΔCT) represent changes normalized to the parental IBC cell. The primers designed to target XIAP and β-actin were described previously (18).

Construction of XIAP IRES luciferase plasmid and transfection of cells

The luciferase construct was constructed by inserting the 5’UTR of XIAP (kindly provided by Dr. Martin Holcik, University of Ottawa) upstream of luciferase in the pGL3 Basic vector (Promega, Madison, WI) and a CMV promoter upstream of the 5’UTR (pGL3-XIAP.IRES). The negative control constructs [pGL3-XIAP.IRES (cont 1) and pGL3-XIAP.IRES (cont 2)] were constructed by deleting the sequence between the PstI (−800-731) and PstI/HindIII (−800-0) restriction sites, respectively. For transfection of DNA, cells were seeded in their respective media in 24-well plates (Corning Incorporated) and allowed to reach 80–90% confluency. At that time, cells were transfected with 1.5 µg pGL3-hUTR.luc and 0.5 µg pRL-TK (Promega) DNA using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s instructions. Cells were incubated for 24 h and lysed for luciferase activity assay.

Luciferase activity assay

Cells were lysed for 15 min in 500 µl luciferase lysis buffer (35 mg/ml Tris base, 5 mg/ml CDTA, 10% glycerol, 0.5% Triton-X 100, pH 7.8) and 25 µl of the lysate was added to a 96-well plate (Greiner Bio-One, Monroe, NC). Luciferase activity was determined using a luminometer (Turner Biosystems, Sunnyvale, CA). Firefly or renilla luciferase substrate [1 mM luciferin or colelentrerazine (Gold Biotechnology, St. Louis, MO) in 15 mM MgSO4, 15 mM K2HPO4, 4 mM EGTA, 1 mM DTT, 0.1 mM ATP] was added (100 µl) to wells and luciferase activity was read after 10 s.

Caspase 9 activity assay

Cells were seeded in 6-well plates (Corning Incorporated), and the next day cells were treated with embelin (50 µM) for 4 h in regular growth media. DMSO (at the same concentration as embelin) was used as a vehicle control. After incubation, caspase 9 activity was determined in 3 µg total cell lysates using the Caspase-Glo® Assay (Promega) as per the manufacturer’s instructions. Peak light intensity of treatment wells was normalized to DMSO.

Nucleosome Enrichment Assay

Cells were seeded in 96-well plates (Corning Incorporated). Embelin (50 µM) and staurosporine (5 µM) were made in regular growth media. DMSO (at the same concentration as drug treatments) was used as a vehicle control. After 20 h incubation, nucleosome enrichment was determined by the Cell Death Detection ELISAPLUS (Roche Applied Science; Mannheim, Germany) as per the manufacturer’s instructions. Nucleosome enrichment was calculated by: (mU sample – Blank)/ (mU untreated – Blank) *100.

Statistical analysis

The statistical analyses were performed using Graphpad InStat Student’s two tailed t-test. Differences were considered significant at p<0.05.

Results

Development of a model of acquired resistance of IBC cells to an ErbB1/2 tyrosine kinase inhibitor

The effect of a laboratory grade lapatinib analog (GW583340) on cell growth and cell death was characterized in two well-established IBC cell lines isolated from primary IBC tumors (37): SUM190 (ErbB2 overexpressing) and SUM149 (ErbB1 activated). Because resistance to lapatinib monotherapy in patients treated with daily doses of lapatinib is commonly seen (31), GW583340-resistant lines (referred to here as rSUM190 and rSUM149) were established by chronic exposure of the parental SUM190 and SUM149 cells to increasing concentrations of GW583340 for greater than 3 months (see Materials and Methods). GW583340 treatment at concentrations in which the rSUM149 and rSUM190 cells were established for acquired resistance to this agent caused significant growth inhibition in the parental SUM149 (Fig. 1A; p<0.005) and SUM190 (Fig. 1B; p<0.05) cells. However, the growth curves indicate that rSUM149 and rSUM190 cells have similar doubling times to their parental counterparts (SUM149 14.1 h vs. rSUM149 14.9 h; SUM190 39.6 h vs. rSUM190 40.1 h). Analysis of cell death as measured by Annexin/PI staining shows an increase in total dead cells 24 h post treatment in the parental cells compared to rSUM149 (Fig. 1C, left panel; p<0.05) and rSUM190 (Fig. 1D, left panel; p<0.05) cells. In addition, exposing rSUM149 and rSUM190 cells (which are maintained in 7.5 µM and 2.5 µM GW583340, respectively) to increasing concentrations of GW583340 (up to 20 µM for 24 h) caused only a modest increase (10–20%) in cell death as measured by 7-AAD viability stain (Fig. 1C, D, right panels, left y-axis) and no decrease in cell proliferation (MTT assay, Fig. 1C, D; right panels, right y-axis) compared to 70–80% cell death and decrease in proliferation in the parental SUM149 and SUM190 cells. It should be noted that the effect of GW583340 on decreasing cell proliferation in parental SUM149 and SUM190 cells saturates to 50–60% at 10–20 µM at 24 h and only increase in time of treatment causes further significant growth inhibition. These data support the establishment of two IBC cell models (rSUM149 and rSUM190) with acquired resistance to GW583340.

Figure 1.

Figure 1

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Effect of GW583340 on cell growth, proliferation, and death in parental and resistant IBC cells. Untreated SUM149 cells were compared to SUM149 and rSUM149 cells treated with 7.5 µM GW583340 (A) and untreated SUM190 cells were compared to SUM190 and rSUM190 cells treated with 2.5 µM GW583340 (B). Cell growth was assessed after 1, 2, 3, and 7 days of continuous exposure to GW583340 by trypan blue exclusion assay. Cell count is presented as the total cell number. [**p<0.005, rSUM149 vs. SUM149 treated with 7.5 µM GW583340 (n=2–3); [*p<0.05, rSUM190 vs. SUM190 treated with 2.5 µM GW583340 (n=2)] Cell death and proliferation of rSUM149 cells and SUM149 cells (C) and rSUM190 and SUM190 cells (D) treated for 24 h with GW583340. Cell death was assessed by Annexin V/PI (left panel) or 7-AAD (right panel, left y-axis) staining and proliferation was determined by MTT assay (right panel, right y-axis). Bars represent the mean ± SEM of the total dead cell count relative to the DMSO control or percentage proliferation relative to the DMSO control. [*p<0.05, **p<0.005, n=2]

Dysregulation of the apoptotic pathway and not inhibition of ErbB signaling contributes to acquired resistance to GW583340

Evaluation of the effect of GW583340 on the ErbB signaling pathways in SUM149 and SUM190 IBC cells revealed that treatment with GW583340 caused a marked and comparable downregulation of p-ErbB1 in both parental SUM149 and rSUM149 cells compared to untreated parental cells (Fig. 2A). Similarly, expression of p-ErbB2, p-AKT, and p-MAPK were inhibited in the GW583340-treated SUM190 and rSUM190 cells. However, an increase in total MAPK expression was observed in the rSUM190 cells (Fig. 2B).

Figure 2.

Figure 2

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A, Immunoblot analysis of SUM149 and rSUM149 cells with an antibody against p-ErbB1. The p-ErbB1 blot was stripped and reprobed for ErbB1 total protein. B, Immunoblot analysis of SUM190 and rSUM190 cells with antibodies against p-ErbB2, p-MAPK, and p-AKT. Phospho blots were stripped and reprobed for corresponding total protein. In addition, GAPDH was used as a loading control for MAPK. C, Immunoblot analysis (left panel) of SUM190 cells treated with 40 µM LY294002 with an antibody against p-AKT. The phospho blot was stripped and reprobed for total AKT. Cell death (right panel) was assessed by Annexin V/PI staining of rSUM190 cells and SUM190 cells treated for 48 h with 40 µM LY294002. DMSO was used as a vehicle control. Bars represent the mean ± SEM of the percentage of total dead cells. [*p<0.05, n=2] Numbers represent densitometric analysis of phospho protein to total protein in all immunoblots.

In order to evaluate the effect of direct inhibition on PI3K (downstream effector in ErbB signaling), SUM190 and rSUM190 cells were treated with a PI3K inhibitor (LY294002). LY294002 treatment inhibited p-AKT as per its mechanism of action and increased cell death of SUM190 cells (Fig. 2C; p<0.05). In contrast, the rSUM190 cells were not affected by direct inhibition of PI3K signaling using LY294002. This insensitivity potentially indicates that in ErbB2 overexpressing rSUM190 cells, GW583340 retains the ability to inhibit p-ErbB2, but the cells no longer rely on signaling downstream of the ErbB2 receptor. These data suggest that the primary mechanism of GW583340 action as a dual ErbB1/2 kinase inhibitor (38) is not compromised in the rSUM149 and rSUM190 cells and thereby does not explain the decreased sensitivity to GW583340-induced cell death observed in the rSUM149 and rSUM190 cells.

The effect of GW583340 on apoptotic signaling was thereby interrogated in these cell lines. Western immunoblot analysis of key anti-apoptotic proteins revealed sustained survivin (Fig. 3A) and Bcl-2 (Fig. 3B) expression in rSUM149 and rSUM190 cells. Further, immunoblot analysis of Forkhead box O3 (FOXO3a), a transcription factor known to promote pro-apoptotic gene transcription, revealed a decrease in FOXO3a expression in rSUM149 and rSUM190 cells (Fig. 3C); however, the difference was more marked in the rSUM190 cells. XIAP, one of the most potent caspase inhibitors that can modulate both the mitochondrial and extrinsic apoptotic signaling cascades, was characterized (Fig. 4) in the IBC cells described herein. A 2–3 fold overexpression of XIAP protein levels was observed by immunoblot analysis in rSUM149 and rSUM190 cells compared to untreated parental cells (Fig. 4A). In addition, inhibition of XIAP expression in SUM149 cells and cleavage (data not shown) were observed post-GW583340 treatment, which was similar to previous results from our lab in SUM190 cells (18). XIAP immunofluorescence (Fig. 4B) and further quantitative analysis of the mean intensity of XIAP staining per pixel supports the XIAP overexpression in rSUM149 and rSUM190 cells (Fig. 4C; SUM149 vs. rSUM149, p<7×10−7; SUM190 vs. rSUM190, p<5×10−6). These data identify a mechanism of apoptotic dysregulation associated with overexpression of XIAP in rSUM149 and rSUM190 cells maintained in GW583340.

Figure 3.

Figure 3

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Effect of acquired resistance to GW583340 on apoptotic signaling in IBC cells. A, Survivin immunoblot analysis of parental and resistant IBC cells. B, Bcl-2 immunoblot analysis of parental and resistant IBC cells. C, FOXO3a immunoblot analysis of parental and resistant IBC cells. GADPH was used as a loading control for all immunoblots.

Figure 4.

Figure 4

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Effect of acquired resistance to GW583340 on XIAP in IBC cells. A, XIAP immunoblot analysis of rSUM190 and rSUM149 cells. GADPH was used as a loading control. Numbers represent densitometric analysis of XIAP normalized to GAPDH. B, Representative fluorescent microscopy images of parental and resistant SUM149 and SUM190 cells probed with an XIAP antibody and counterstained with Hoechst. C, Mean XIAP staining intensity per pixel in parental and resistant IBC cells. Bars represent the average mean XIAP staining intensity per pixel ± SEM in over 20 single cells taken from 10 different fields. D, Effect of exogenous, stable XIAP overexpression on viability post-GW583340 treatment. Wildtype XIAP (wtXIAP) or vector control (Vector cont.) were stably overexpressed in parental SUM149 cells (left panel, immunoblot analysis). Actin was used as a loading control, and numbers represent densitometric analysis of XIAP normalized to actin. Cells were treated for 24 h with GW583340 and viability was assessed via trypan blue exclusion (left panel). Bars represent mean +/− SEM. (*p<0.05, n=2).

To determine whether XIAP overexpression is a potential factor in acquired resistance to GW583340, parental SUM149 cells were stably transfected using a lentiviral expression system with exogenous wildtype XIAP and a corresponding vector control as described in Materials and Methods (Fig. 4D, left panel). Data in Fig. 4D show that exogenous overexpression of XIAP in parental IBC cells reverses sensitivity of parental SUM149 cells to GW583340-induced cell death, which demonstrates that XIAP expression is sufficient to mediate resistance of these cells to the ErbB1/2 tyrosine kinase inhibitor.

XIAP overexpression in IBC cells with acquired resistance to GW583340 is driven by IRES-mediated translation

To address the mechanism of XIAP upregulation in the GW583340-resistant IBC cells, XIAP transcription and translation of XIAP protein were characterized in the IBC cells. Real time RT-PCR analysis (Fig. 5A) showed no significant change in XIAP mRNA levels (SUM149 vs. rSUM149, p=0.467; SUM190 vs. rSUM190, p=0.233). XIAP has been identified to have an IRES (internal ribosomal entry sequence) element in its 5’UTR that can be used as a non-canonical translational start site in times of cellular stress (8–10). In order to characterize the IRES-mediated translation of XIAP in the IBC cells, a luciferase reporter construct was generated wherein the 5’UTR of XIAP, which contains the IRES, was cloned immediately upstream of the firefly luciferase gene (pGL3-XIAP.IRES). Data in Fig. 5B reveal that both rSUM149 and rSUM190 cells had higher luciferase activity than their parental counterparts when firefly luciferase expression was normalized to the co-transfected renilla luciferase plasmid (SUM149 vs. rSUM149, p<0.005; SUM190 vs. rSUM190, p<0.005). Additionally, transfection of cells with truncated forms of the XIAP IRES [pGL3-XIAP.IRES (cont 1) and pGL3-XIAP.IRES (cont 2)], which acted as negative controls, did not elicit any luciferase activity. These data demonstrate that the upregulation of XIAP in GW583340-resistant cells is predominantly driven by IRES-mediated translation of XIAP and not increase in XIAP mRNA.

Figure 5.

Figure 5

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Effect of acquired resistance to GW583340 on XIAP mRNA expression and protein translation in IBC cells. A, RT-PCR analysis of XIAP mRNA expression in parental and resistant IBC cells. β-actin was used as an internal control. [p= ns (not significant), n=2] B, Luciferase activity was quantitated in SUM149, rSUM149, SUM190, and rSUM190 cells co-transfected with pGL3-Basic, pGL3-XIAP.IRES, pGL3-XIAP.IRES(cont 1), or pGL3-XIAP.IRES(cont 2) and a renilla plasmid (pRL-TK). Numbers represent the ratio of firefly luciferase activity to renilla luciferase activity taken as a percentage of their respective untreated readout. [** p<0.005, n=2]

Inhibition of XIAP function using a small molecule inhibitor causes apoptosis and overcomes GW583340 resistance

Translational upregulation of XIAP seems to correlate with acquired resistance to GW583340-induced apoptotic response when cells are chronically exposed to GW583340 (Fig. 5B). Additionally, exogenous overexpression of XIAP caused parental IBC cells to be resistant to the cell death induced by GW583340 (Fig. 4D), which is similar to that seen is the acquired resistance model (rSUM149) with endogenously high levels of XIAP (Fig. 4A,B). Therefore, we evaluated the effect of inhibition of XIAP action. For this purpose, embelin (a small molecular inhibitor that has been shown to prevent binding of XIAP to procaspase 9 and thereby increase caspase 9 activity) was employed (34). The mechanism of embelin is demonstrated in Fig. 6A wherein decreased levels of procaspase 9 were observed with increasing concentrations in parental and rSUM149 and rSUM190 cells. This decrease in procaspase 9 post-embelin treatment correlated with increased caspase 9 activity (Fig. 6A, p<0.005) and apoptosis as measured by nucleosome enrichment (Fig. 6A; SUM149 and rSUM149, p<0.005; rSUM149, p<0.05). Treatment of another IBC-like cell line (SUM44) with embelin did not decrease procaspase 9 expression (Fig. 6A), and these cells have been observed to be resistant to the apoptotic-inducing effects of the XIAP inhibitor (data not shown), which demonstrates the specificity of embelin.

Figure 6.

Figure 6

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Effect of embelin, GW583340 alone and in combination on cell death. A, Effect of embelin on procaspase 9 (left panel, immunoblot analysis) and right panel (caspase 9 activity and nucleosome enrichment). GAPDH was used as a loading control for the immunoblots. Numbers represent densitometric analysis of procaspase 9 normalized to GAPDH. Bars represent mean +/− SEM of triplicate values (n=2). B, Effect of GW583340 and embelin treatments as shown by + and − on total cell death in SUM149 and rSUM149 (growing in GW583340) (left panel) and SUM190 and rSUM190 (growing in GW583340) (right panel) cells. Bars represent mean +/− SEM of triplicate values. [resistant cells treated with GW583340 vs. embelin, p<0.05, n=2–3]

Since rSUM149 and rSUM190 cells maintained in GW583340 show XIAP overexpression and are resistant to GW583340-mediated apoptosis, experiments were conducted to determine if inhibition of XIAP action using embelin would sensitize the resistant cells to GW583340. Addition of embelin to the rSUM149 and rSUM190 cells growing in GW583340 for 48 h caused significant cell death compared to GW583340 alone in the absence of embelin (p<0.005). Additionally, treatment of parental cells with both GW583340 (Fig. 1, Fig. 6B) and embelin (Fig. 6A – B) alone or in combination (Fig. 6B) significantly increased cell death compared to vehicle control cells (SUM149, p<0.005; SUM190, p<0.005). Data from our previous study (18) showed that treatment of sensitive parental cells with GW583340 decreases XIAP expression. Taken together, these data indicate that XIAP is a point of failure in both parental and GW583340-resistant IBC cells. Moreover, no synergy was observed between GW583340 and embelin treatment, which is most likely because these drugs affect the same pathway (i.e., decrease in XIAP). In summary, these data demonstrate that inhibition of XIAP binding to procaspase 9 using embelin and resultant increase in caspase activity causes apoptosis and potentially overcomes the acquired resistance to cell death in rSUM149 and rSUM190 cells.

Discussion

We report herein apoptotic dysregulation correlating with XIAP overexpression in two IBC cell models of acquired resistance to a lapatinib analog (GW583340). The parental cells, SUM190 (ErbB2 overexpressing) and SUM149 (ErbB1 activated) derived from primary tumors of IBC patients (37), were sensitive to GW583340-mediated cell death. A marked decrease in p-ErbB2 or p-ErbB1 and corresponding inhibition of downstream signaling were evident in cells with acquired resistance to GW583340 (rSUM190 and rSUM149, respectively), similar to the parental counterparts treated with the drug, suggesting that the primary mechanism of action of GW583340, a dual ErbB1/2 tyrosine kinase inhibitor, was not compromised in the resistant cells.

Lapatinib is a dual tyrosine kinase inhibitor and is therefore effective in tumors with either ErbB2 expression or ErbB1 expression. Both ErbB2 overexpressing and ErbB1 activated IBC cells were sensitive to the growth-inhibitory and apoptotic-inducing effects of GW583340 (a lapatinib analog). Evidence from the clinic has shown that IBC tumors are relatively more responsive to lapatinib than other breast cancer types (RR = 50% in IBC vs. <10% in non-IBC; (29, 30)); however, the response to lapatinib is often short-lived and resistance is common (31). Two recent studies (25, 32) have shown that apoptotic signaling is an important mechanism of lapatinib resistance, and the apoptotic pathways have been characterized to be dysregulated in IBC vs. other LABC types (39–42). Xia et al. (25) reported that acquired resistance to lapatinib in the estrogen receptor (ER)-dependent non-IBC BT474 cells is due to increased activity of the transcription factor FOXO3a, which regulates ER downstream anti-apoptotic proteins such as survivin and Bcl-2. The other report demonstrated that MCL-1 (an anti-apoptotic member of the Bcl-2 family) is increased in colon cancer cells resistant to lapatinib (32). These studies support the idea that dysregulation of the apoptotic signaling pathway plays a key role in the resistance of cancer cells to lapatinib. In addition, a previous study in our lab has shown that XIAP expression correlates with resistance to trastuzumab in the ErbB2 overexpressing SUM190 IBC cells (18) further supporting the hypothesis that the anti-apoptotic signaling pathway is dysregulated in response to ErbB2 targeting agents.

In the present study, a model of acquired resistance to a dual ErB1/2 tyrosine kinase inhibitor (lapatinib analog, GW583340) was generated because resistance to lapatinib monotherapy in patients treated with daily doses of lapatinib is commonly seen (31). The GW583340-resistant lines (rSUM190 and rSUM149) were established by chronic exposure of the parental SUM190 and SUM149 cells to the drug for greater than 3 months. It was shown that the primary mechanism of action of the tyrosine kinase inhibitor remained intact in the GW583340-resistant cellular model, and therefore we hypothesized that the apoptotic pathway was dysregulated. A significant overexpression of XIAP was observed to be mediated by IRES-dependent translation in the acquired resistant cell models studied here. In addition, it has been reported that FOXO3a, a transcription factor that promotes pro-apoptotic gene transcription (43), and XIAP expression show an inverse correlation (43). FOXO3a is upregulated by c-jun N-terminal kinase (JNK) (43), which is negatively regulated by XIAP (44). It is therefore not surprising that FOXO3a expression was decreased in the GW583340-resistant IBC cells wherein XIAP was dramatically upregulated. In addition, rSUM149 and rSUM190 cells had sustained Bcl-2 and survivin levels similar to a previous report in a non-IBC cell line resistant to lapatinib (25).

The present data show that XIAP was specifically overexpressed in the acquired resistance IBC model (rSUM149 and rSUM190). Alternatively, exogenous overexpression of XIAP in parental cells reversed the sensitivity to GW583340-mediated apoptosis, revealing the critical role of XIAP in therapeutic resistance. Interestingly, we observed that in contrast to targeting XIAP, siRNA-mediated inhibition of another IAP (survivin) implicated in therapeutic resistance in breast cancer in these cells had no significant effect on viability or apoptosis (data not shown), similar to our previous report in trastuzumab resistance in IBC (18). This is consistent with the role of survivin as a non-traditional inhibitor of apoptosis as it has not been effectively shown to functionally inhibit caspases (45) but is rather a mitotic regulator (46). Interestingly, XIAP has been previously shown to bind and regulate the function of survivin (47), and therefore it is appealing to speculate that inhibition of both XIAP and survivin may be even more potent than inhibition of these molecules separately.

Embelin, an inhibitor of XIAP’s primary role of a caspase inhibitor (34), was used as a proof of principle agent to cause specific abrogation of the inhibitory interaction between XIAP and procaspase 9; treatment of rSUM190 and rSUM149 cells with embelin decreased cell viability and increased apoptosis. This indicates that XIAP is critical for survival of cells with acquired resistant to GW583340.

It is clear that apoptotic dysregulation is a critical factor in acquired lapatinib resistance in breast cancer. In addition, this study is the first to elucidate that XIAP overexpression corresponding with resistance to GW583340-induced apoptosis in the ErbB2 overexpressing and ErbB1 activated IBC cellular models is not due to increase in XIAP transcription but rather due to increased translation of XIAP via its IRES element present in its 5’UTR (8–10). These unique secondary structures can be used as non-canonical translation start sites during times of cellular stress when traditional protein translation is shut down (7), identifying XIAP as a stress-related target for therapeutic intervention and establishing the feasibility of targeting XIAP in combination with lapatinib to enhance tumor apoptosis in IBC therapy.

Acknowledgements

The authors would like to thank the Duke Light Microscopy Facility for their expertise and advice, Dr. Mark Dewhirst and Dr. Michael Datto for their help in reviewing the manuscript, and Dr. Zachary Hartman for technical help for the cloning experiments. The authors would also like to thank Dr. Martin Holcik for the XIAP IRES plasmids and Dr. Colin Duckett for the XIAP lentiviral plasmids.

This work was supported by funding from American Cancer Society RSG-08-290-01-CCE (GRD), Department of Defense Predoctoral award, W81XWH-08-1-0363 (KMA) and SPORE in breast cancer grant (5P50-CA068438) at Duke Comprehensive Cancer Center.

Abbreviations

BIR

baculoviral IAP repeat

ER

estrogen receptor

IAP

inhibitor of apoptosis protein

IBC

inflammatory breast cancer

IRES

internal ribosomal entry sequence

JNK

c-Jun N-terminal kinase

LABC

locally advanced breast cancer

MAPK

mitogen activated protein kinase

NF-κB

nuclear factor kappa B

UTR

untranslated region

XIAP

X-linked inhibitor of apoptosis protein

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