X-linked inhibitor of apoptosis protein inhibits apoptosis in inflammatory breast cancer cells with acquired resistance to an ErbB1/2 tyrosine kinase inhibitor

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 analogue, ErbB1/2 inhibitor)-resistant IBC cells, we identified the overexpression of an antiapoptotic protein, X-linked inhibitor of apoptosis 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 that 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, showing 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.

Figure 1

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]

Figure 2

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.

Figure 3

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

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).

Figure 5

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]

Figure 6

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]