Smac mimetic Birinapant induces apoptosis and enhances TRAIL potency in inflammatory breast cancer cells in an IAP-dependent and TNF-a-independent mechanism
Jennifer L. Allensworth • Scott J. Sauer •
H. Kim Lyerly • Michael A. Morse •
Gayathri R. Devi
Received: 12 July 2012 / Accepted: 21 November 2012
© Springer Science+Business Media New York 2012
Abstract
X-linked inhibitor of apoptosis protein (XIAP), the most potent mammalian caspase inhibitor, has been associated with acquired therapeutic resistance in inflam- matory breast cancer (IBC), an aggressive subset of breast cancer with an extremely poor survival rate. The second mitochondria-derived activator of caspases (Smac) protein is a potent antagonist of IAP proteins and the basis for the development of Smac mimetic drugs. Here, we report for the first time that bivalent Smac mimetic Birinapant induces cell death as a single agent in TRAIL-insensitive SUM190 (ErbB2-overexpressing) cells and significantly increases potency of TRAIL-induced apoptosis in TRAIL- sensitive SUM149 (triple-negative, EGFR-activated) cells, two patient tumor-derived IBC models. Birinapant has high binding affinity (nM range) for cIAP1/2 and XIAP. Using isogenic SUM149- and SUM190-derived cells with dif- ferential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) and another bivalent Smac mimetic (GT13402) with high cIAP1/2 but low XIAP binding affinity (Kd [ 1 lM), we show that XIAP inhibition is necessary for increasing TRAIL potency. In contrast, single agent efficacy of Birinapant is due to pan-IAP antagonism. Birinapant caused rapid cIAP1 degradation, caspase acti- vation, PARP cleavage, and NF-jB activation. A modest increase in TNF-a production was seen in SUM190 cells following Birinapant treatment, but no increase occurred in SUM149 cells. Exogenous TNF-a addition did not increase Birinapant efficacy. Neutralizing antibodies against TNF-a or TNFR1 knockdown did not reverse cell death. However, pan-caspase inhibitor Q-VD-OPh reversed Birinapant- mediated cell death. In addition, Birinapant in combination or as a single agent decreased colony formation and anchorage-independent growth potential of IBC cells. By demonstrating that Birinapant primes cancer cells for death in an IAP-dependent manner, these findings support the development of Smac mimetics for IBC treatment.
Keywords : Smac/DIABLO · Inhibitor of apoptosis protein (IAP) · TRAIL · Inflammatory breast cancer (IBC) · TNF- a
Introduction
Acquired therapeutic resistance due to tumor cell adapta- tion to persistent therapeutic stress is an unmet challenge in inflammatory breast cancer (IBC), one of the most lethal subtypes of breast cancer [1]. We have identified X-linked inhibitor of apoptosis protein (XIAP) overexpression via post-transcriptional mechanisms as a dominant feature of apoptotic dysregulation involved in acquired therapeutic resistance to epidermal growth factor receptor (EGFR) and/or ErbB2/HER2-targeting drugs like trastuzumab and lapatinib as well as apoptosis-inducing agents such as TNF- related apoptosis-inducing ligand (TRAIL) [2–5]. This is supported by other studies that have shown consistent differences in molecular phenotype between IBC and locally advanced non-IBC tumors, with IBC characterized by a hyperproliferative state and apoptotic dysregulation [2–4, 6–13]. Thus, the development of novel therapeutic strategies targeting the anti-apoptotic signaling pathway is highly relevant in IBC.
XIAP, the most potent caspase inhibitor, is a member of the highly conserved inhibitor of apoptosis protein (IAP) family of endogenous apoptosis inhibitors. IAPs have been shown to inhibit apoptosis induced by both extrinsic (e.g., death receptor) and intrinsic (e.g., DNA damage) stimuli. They are characterized by the presence of 1–3 baculoviral IAP repeat (BIR) domains which mediate interactions with other proteins. Cellular IAP 1 and 2 (cIAP1 and 2) were originally identified as components of the TNF-a receptor (TNFR2) complex and are increasingly recognized as modulators of diverse extrinsic signals. They possess a C-terminal RING (Really Interesting New Gene) domain which has E3 ubiquitin ligase activity. By maintaining the ubiquitination states of key substrates such as RIP kinases and caspases, they are able to promote survival signaling. XIAP, in addition to harboring a C-terminal RING domain with E3 ligase activity, is a direct caspase inhibitor which can bind to activated caspases-3, -7, and -9 and inhibit their proteolytic activity [14].
While XIAP is not considered to be a classical onco- gene, it has been detected at elevated levels in tumors resistant to chemotherapy and is associated with poor outcome in several cancers [15–18]. Two broad approaches have been taken to develop clinical inhibitors of XIAP, including antisense oligonucleotides that reduce XIAP expression and small molecule inhibitors that antagonize XIAP’s caspase inhibitory function. These molecules are currently being evaluated in preclinical and clinical phase I–II studies [19, 20]. One class of XIAP inhibitory mole- cules being developed as anticancer therapeutic agents is second mitochondria-derived activator of caspases (Smac) mimetics, which are modeled after the AVPI tetrapeptide IAP binding motif (IBM) of Smac/DIABLO [21]. Smac mimetics are attractive since Smac/DIABLO is considered to be one of the main antagonists of XIAP and other IAPs because it can bind to the BIR3 region of IAP proteins through its N-terminal AVPI tetrapeptide. In addition, XIAP overexpression can inhibit the release of endogenous Smac from the mitochondria [22]. Recently, expression of Smac/DIABLO in 62 breast cancer patient specimens assessed by flow cytometry was shown to inversely cor- relate with tumor stage [23]. Smac mimetics are potent pro- apoptotic agents, and their mechanisms include: (1) steri- cally and/or competitively occluding the binding sites of XIAP from caspases-3, -7, and -9; (2) XIAP degradation; (3) TNF-a-mediated cell death; and (4) inhibition and degradation of other IAP family members. This is impor- tant due to the fact that in many cases when XIAP is tar- geted, other IAPs suppress apoptosis by compensatory mechanisms [24–28]. Currently, Smac mimetics including Birinapant (formerly called TL32711) are in clinical trials [29]. While only a subset of tumor cell lines is sensitive to Smac mimetics as single agents, combining Smac mimetics with different therapeutic agents has been shown to increase potency of those agents [30–35]. The predominant mechanism of many of the Smac mimetic agents seems to be TNF-a dependent [28, 36]. In this study, we demon- strate that the mechanism of action of Birinapant as a single agent and in combination with TRAIL is not dependent on TNF-a, but is predominantly due to IAP downregulation and caspase activation in the IBC cellular models.
Materials and methods
Cell lines and reagents
SUM149 and SUM190 IBC cell lines were obtained from Asterand, Inc. (Detroit, MI) and cultured as described previously [3]. Asterand characterizes cell lines using short tandem repeat polymorphism analysis. Cells were banked upon receipt and cultured for no more than 6 months before use in this study. SUM149 cells stably expressing wtXIAP and FG9 vector control were generated and maintained as described previously [4]. TRAIL was pur- chased from Enzo Life Sciences (Farmingdale, NY), and TNF-a was purchased from Sigma Aldrich (St. Louis, MO). A TNF-a neutralizing antibody was purchased from R&D Systems (Minneapolis, MN), and Q-VD-OPh pan- caspase inhibitor was purchased from Cal Biochem (Billerica, MA). TNFR1 siRNA, control scramble siRNA (Thermo/Dharmacon On-Target plus SMART pool reagents: Control siRNA #L-004445-00-0005, TNFRSF1A #L-005197-00-0005), and Dharmafect transfection reagent were purchased from Thermo Scientific (Waltham, MA). Smac mimetics Birinapant and GT13402 were obtained from TetraLogic Pharmaceuticals (Malvern, PA) and have been designed based on the four N-terminal amino acids of Smac/DIABLO (Ala-Val-Pro-Ile) and caspase-9 (Ala-Thr- Pro-Phe) [29].
Generation of XIAP-knockdown IBC cell line
SUM190 cells stably expressing shRNA against XIAP and FG12 vector control were generated using a lentiviral expression system (kindly provided by Dr. Colin Duckett, University of Michigan, Ann Arbor, MI). In brief, HEK293T cells were transfected using polyethylenimine with 5 lg of pHCMV, pRRE, and pRSVrev [37], which drive the expression of lentiviral structural proteins, and 5 lg of pFG12 shXIAP GFP or pFG12 GFP DNA; 24 h post-transfection, media was changed. After 40 h post- transfection, the virus-containing media was collected from the HEK293T cells and filtered through a 0.45 mm Millex HV PVDF filter unit (Millipore, Billerica, MA) onto cells with 25 mM polybrene (Sigma Aldrich). After 4 h, fresh media was added, and cells were incubated for an addi- tional 48 h at 37 °C, 5 % CO2. Stable shXIAP and FG12 cells were sorted using FACS for high GFP expression.
Fluorescence polarization assay
The binding affinities of compounds to XIAP and cIAP1 were determined as described previously [38] using a fluo- rogenic substrate and are reported as Kd values. Initially, the dissociation constant (Kd) for the fluorescently labeled modified Smac peptide (AbuRPF-K(5-Fam)-NH2; FP pep- tide) was determined using a fixed concentration of peptide (5 nM) and titrating varying concentrations of protein (0.075–5 lM in half log dilutions). The dose–response curves were produced by a nonlinear least squares fit to a single-site binding model using GraphPad Prism (Graphpad Software, La Jolla, CA; data not shown), with 5 nM of FP peptide and 50 nM of XIAP used in the assay. Various concentrations of Smac mimetics (100–0.001 lM in half log dilutions) were added to FP peptide:protein binary complex for 15 min at room temperature in 100 lL of 0.1 M potas- sium phosphate buffer, pH 7.5, containing 100 mg mL-1 bovine c-globulin. Following incubation, the polarization values were measured on a Perkin-Elmer Victor2V multi- label plate reader (Waltham, MA) using a 485 nm excitation filter and a 520 nm emission filter. IC50 values were deter- mined from the plot using nonlinear least squares analysis in Graphpad Prism. Calculations were based on a maximum signal (protein BIR:FP peptide complex treated with DMSO alone) after subtraction of background.
Immunoblot analysis
Western immunoblot analysis was carried out as described previously [2]. Cells were harvested at 4 or 24 h post treatment. Membranes were incubated with primary anti- bodies for XIAP (BD Biosciences, San Jose, CA, 1:1,000), DR4, PARP (1:1,000), actin, and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, 1:4,000), cIAP1 and cIAP2 (R&D Systems, 1:1,000), caspase-8, JNK, p-JNK, NF-jB (p65), p-NF-jB (p65), TNFR1, and caspase-3 (Cell Signaling Technologies, Danvers, MA, 1:1,000) at 4 °C overnight. Membranes were washed and incubated with anti-mouse or anti-rabbit HRP-conjugated antibodies (Cell Signaling Technologies) for 1 h at room temperature. Chemiluminescent substrate (Thermo Scientific) was applied for 5 min, and membranes were exposed to film. Densitometric analysis was performed using NIH ImageJ software [39].
Cell viability
Trypan blue exclusion assay was performed as described previously [2]. Cells were seeded in 6 well plates at 7.5 9 104 (SUM149) or 1.5 9 105 (SUM190) cells per well and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL-1), Birinapant (0–10,000 nM), GT13402 (0–10,000 nM), TNF-a (50 ng mL-1), TNF-a neutralizing antibody (10 lg mL-1), pan-caspase inhibitor Q-VD-OPh (20 lM), or a combination as indicated. All treatments were applied for 24 h, and then the cells were trypsinized and resuspended in PBS. Next, 10 lL of cell suspension was added to 10 lL 0.4 % trypan blue, and 10 lL of the mixture was loaded onto a hemocytometer; cells were counted, and live and dead cell numbers were recorded.
Clonogenic growth assay
Cells were plated in triplicate in 6 well plates at 250–500 cells per well (SUM149) or 500–1,000 cells per well (SUM190) and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL-1), Birinapant (0–10,000 nM), GT13402 (0–10,000 nM), TNF-a (50 ng mL-1), TNF-a neutralizing antibody (10 lg mL-1), pan-caspase inhibitor Q-VD-OPh (20 lM), or a combination as indicated. After 24 h treatments, the cells were washed twice with PBS, and regular growth media was added. The cells were then allowed to grow for 5–14 days, changing the media every 4–5 days. Once colonies of at least 50 cells were observed, the cells were washed with PBS, fixed, stained with 0.4 % crystal violet, then rinsed in cold water and left to dry overnight. Colonies were counted and imaged using a ColCount (Oxford Optronix, Oxford, UK), and colonies formed per cells plated was calculated. Numbers were normalized to untreated.
Anchorage-independent growth (AIG) assay
Cells were plated in 6 well plates at 7.5 9 104 (SUM149) or 1.5 9 105 (SUM190) cells per well and allowed to adhere overnight. Treatments were applied for 24 h, after which cells were harvested and counted with trypan blue viability stain. A base layer of 0.7 % agarose in regular growth medium was poured into wells of a 12 well plate and allowed to solidify at 4 °C. Then, 1.25 9 104 cells per well from each treatment were plated in triplicate in 0.45 % agarose in regular growth medium on top of the base layer and allowed to solidify at 4 °C. Plates were then transferred to a 37 °C incubator with 5 % CO2 and allowed to grow for 14–21 days. Once visible colonies had formed, they were counted under a microscope, and colony counts were nor- malized to the untreated sample. Images of representative fields were taken with 59 magnification using a Zeiss Axio Observer A1 microscope (Thornwood, NY), Hamamatsu Orca ER digital camera (Bridgewater, NJ), and MetaMorph software (Molecular Devices, Sunnyvale, CA).
Annexin-V staining
Cells were seeded in 6 well plates at 7.5 9 104 (SUM149) or 1.5 9 105 (SUM190) cells per well and allowed to adhere overnight. Cells were treated with TRAIL (0–100 ng mL-1), Birinapant (0–1,000 nM), GT13402 (0–1,000 nM), or a combination as indicated for 12 h. Cells were harvested with 0.25 % trypsin (-EDTA), washed with PBS, and resuspended in biotin-conjugated Annexin-V (Beckman Coulter, Brea, CA) for 5 min at RT. Cells were washed again with PBS and resuspended in streptavidin-conjugated FITC (Life Technologies, Grand Island, NY) and 7-AAD (BD Pharminogen, San Jose, CA) dyes for 15 min on ice. Cells were washed and resus- pended in PBS, and then at least 25,000 events were col- lected on a BD FACSCalibur flow cytometer. Results were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
TNF-a measurement
TNF-a protein levels were measured in cell culture supernatants using the BD OptEIA TNF-a ELISA kit II according to the manufacturer’s instructions. In brief, 200 lL of culture supernatants were tested in triplicate. A standard curve was generated using the manufacturer supplied standard. Absorbance was read at 450 nm on a Perkin-Elmer Victor2V multi-label plate reader. The 570 nm background correction was subtracted from 450 nm values to yield the final results.
TNFR1 knockdown
Cells were seeded in 6 well plates at 1.5 9 105 cells per well and allowed to adhere overnight. After 24 h, either scramble control siRNA or TNFR1 targeting siRNA at 100 nM was applied in the presence of Dharmafect transfection reagent. Birinapant (0–1,000 nM) was added the day after transfection, and cells were harvested after 24 h for trypan blue viability staining and western immunoblotting to confirm knockdown.
Statistical analysis
Statistical analyses were performed using Graphpad Prism Version 4 and the student’s two-tailed t test. Differences were considered significant at P \ 0.05.
Results
IAP binding affinity of novel bivalent Smac mimetics
A fluorescence polarization assay was used to determine the dissociation constants (Kd) of Smac mimetics Birina- pant and GT13402 for XIAP and cIAP1 by monitoring the decrease in fluorescence polarization signal due to com- petitive displacement of an FP peptide from the BIR3/FP peptide binary complex. Both Smac mimetic compounds induced a dose-dependent decrease in the fluorescence signal. As shown in Table 1, the binding constants (Kd) of Birinapant for XIAP and cIAP1 were determined to be 45 and \1nM, respectively. In contrast, GT13402 had lower affinity for XIAP with the Kd close to 1 lM but similar affinity for cIAP1 as Birinapant (Kd \ 1 nM).
Smac mimetic enhances TRAIL potency in a TRAIL- sensitive IBC cell line by cIAP1/2 degradation and caspase-8 and PARP cleavage
The basal-type IBC cell line SUM149 has been observed to be TRAIL-sensitive [40, 41], which was supported by data in Fig. 1a displaying a dose-dependent decrease in viability following TRAIL treatment (0–100 ng mL-1). Character- ization of the Smac mimetic compounds as single agents in SUM149 cells revealed that neither Birinapant nor GT13402 is effective in inducing cell death as measured by trypan blue exclusion assay, with *95 % of cells still viable following treatment with the highest concentrations tested (1,000 nM Birinapant, 10,000 nM GT13402—Fig. 1a). However, combinations of TRAIL (10–50 ng mL-1) and Birinapant (0–1,000 nM) enhanced TRAIL potency in SUM149 cells, with a statistically significant *25 % reduction in viability achieved with as low as 25 ng mL-1 TRAIL + 30 nM Birinapant (Fig. 1b). GT13402, which has low affinity for XIAP compared to Birinapant, did not significantly enhance TRAIL potency, even at the highest concentration (10,000 nM) tested (Fig. 1b). Birinapant as a single agent caused significant degradation of cIAP1 and 2, which was not enhanced by the addition of TRAIL (Fig. 1c). While a slight decrease in XIAP expression can be seen following TRAIL treatment, no significant decrease in XIAP levels was observed following combinatorial treatments (Fig. 1c). However, Birinapant + TRAIL-treated cells showed increased levels of active caspase-8 and caspase-3, as well as PARP cleavage, over single agents within 4 h of treatment, indicative of apoptosis-mediated cell death (Fig. 1c). Annexin-V/7-AAD flow cytometric staining further con- firmed the mechanism of death to be apoptosis, with a dose- dependent increase in cells staining positive for Annexin-V (white + gray bar) from *30 % with TRAIL alone to 50–55 % following treatment with Birinapant + TRAIL after 12 h (Fig. 1d).
Because XIAP overexpression has been identified as a critical factor in TRAIL resistance [15, 42, 43], we wanted to delineate the role of XIAP expression in sensitivity to Smac mimetics. To do this, we characterized both Birina- pant and GT13402, which have differential affinity for XIAP, in SUM149 wtXIAP, a SUM149-derived isogenic cell line with stable exogenous XIAP overexpression [4]. Data in Fig. 1e show that Birinapant was more effective in increasing TRAIL potency than GT13402 in SUM149 wtXIAP. This reveals the IAP specificity of the Smac mimetics and the importance of targeting XIAP along with cIAP1 and 2 in this model.
Smac mimetics induce apoptosis as single agents in a dose-dependent manner in TRAIL-resistant SUM190 IBC cells
In order to evaluate the efficacy of the Smac mimetics in a TRAIL-resistant cell line, we characterized Birinapant and GT13402 in SUM190, a cell line isolated from a patient IBC tumor with ErbB2-overexpression and insignificant DR4 expression (a factor that can contribute to TRAIL resistance) as shown in Fig. 2a. SUM190 cells were only moderately sensitized to TRAIL (Fig. 2b, graph) when XIAP was stably downregulated using an XIAP-targeting shRNA lentiviral construct (SUM190 shXIAP, Fig. 2b, immunoblot).
Fig. 1 Smac mimetics sensitize SUM149 cells to TRAIL-mediated cell death. a Viability as determined by trypan blue exclusion assay of SUM149 cells treated with Birinapant (0–1,000 nM), GT13402 (0–10,000 nM), and TRAIL (0–100 ng mL-1) alone. Bars represent mean ± SEM viable cells taken as a percentage of total cells (n = 2–3; *P \ 0.05, #P \ 0.005, all comparisons made to untreated). b Viability as determined by trypan blue exclusion assay of SUM149 cells treated with TRAIL (10–50 ng mL-1) alone or in combination with Birinapant (0–1,000 nM) or GT13402 (0–10,000 nM). Bars represent mean ± SEM viable cells taken as a percentage of total cells (n = 3–5; *P \ 0.05, #P \ 0.005, all comparisons made between TRAIL + Smac mimetic and same concentration of TRAIL alone). c Western immunoblot analysis of cIAP1, cIAP2, XIAP, caspase-3, caspase-8, and PARP expression in SUM149 cells treated with Birinapant (0–1,000 nM) ± TRAIL (50 ng mL-1). GAPDH was used as a loading control. d Annexin- V/7-AAD flow cytometric staining of SUM149 cells treated with Birinapant (0–1,000 nM) ± TRAIL (50 ng mL-1) (n = 2). e Viabil- ity as determined by trypan blue exclusion assay of SUM149 wtXIAP cells treated with TRAIL (50 ng mL-1) and Birinapant or GT13402 (0–1,000 nM). Bars represent mean ± SEM viable cells taken as a percentage of total cells (n = 2–3; *P \ 0.05, #P \ 0.005, all comparisons made to untreated).
In contrast, Birinapant, which targets cIAP1/2 and XIAP with high affinity, significantly decreased the viability of SUM190 cells in a dose-dependent manner as a single agent. Data in Fig. 2c, top panel show *50 % cell death at 300 nM as measured by trypan blue exclusion assay. The requirement of pan-IAP antagonism is supported by the observations (Fig. 2c): (1) treatment with GT13402 (lower affinity for XIAP) was also effective as a single agent in inhibiting SUM190 cell viability; (2) Birinapant (high affinity for cIAP1/2 and XIAP) is more potent (20 % via- bility at 1,000 nM) than GT13402 (60 % viability at 1,000 nM) in this viability assay; (3) Birinapant treatment in the XIAP knockdown cell line (SUM190 shXIAP) caused an overall reduction in viability at lower doses (30–300 nM) compared to the parental SUM190 cells at the corresponding doses; (4) the difference in potency between the two compounds with differential affinity for XIAP was attenuated in the XIAP knockdown cell line (Fig. 2c, lower panel).
To confirm apoptosis with Birinapant treatment, immunoblot analysis was conducted, and data in Fig. 2d showed a significant decrease in cIAP1 levels and enhanced PARP cleavage. However, no decrease in XIAP expression or JNK phosphorylation levels was observed (Fig. 2d). In addition, Annexin-V/7-AAD staining by flow cytometric analysis was carried out at an early time point (12 h). Data in Fig. 2e show an increase in Annexin-V positive cells (white + gray bar) with no change in 7-AAD single positive cells (black bar), further supporting an apoptotic mechanism of cell death.
Fig. 2 Pan-IAP antagonism is important for Smac mimetic-induced apoptosis in SUM190 cells. a Western immunoblot analysis showing TRAIL receptor DR4 expression in SUM190 and SUM149 cells. GAPDH was used as a loading control. b Graph, viability as determined by trypan blue exclusion assay of SUM149, SUM190, and SUM190 shXIAP after treatment with TRAIL (0–100 ng mL-1) at 24 h. Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 3–5; #P \ 0.005, all comparisons made between SUM190 and SUM190 shXIAP at the same concentration of TRAIL). Immunoblot, XIAP expression in SUM190 FG12 vector control and SUM190 shXIAP cells. Actin was used as a loading control. c Top panel SUM190 cell viability as determined by trypan blue exclusion assay when treated with Birinapant or GT13402 (0–10,000 nM). Lower panel, Viability of SUM190 shXIAP cells as determined by trypan blue exclusion assay treated with Birinapant or GT13402 (0–10,000 nM). Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 3–5; *P \ 0.05, all comparisons made between Birinapant and GT13402 at the same concentration). d Western immunoblot analysis of cIAP1, XIAP, and PARP expression, and JNK phosphorylation status in SUM190 cells treated with Birinapant (0–1,000 nM) for 4 h. GAPDH and total JNK were used as loading control. e Annexin-V/7-AAD flow cytometric staining of SUM190 cells treated with Birinapant (B; 0–1,000 nM) or TRAIL (100 ng mL-1) (n = 2; #P \ 0.005; all comparisons made to the untreated sample based on total Annexin-V staining). f Western immunoblot analysis of total NF-jB and phosphorylation status in SUM190 cells treated with Birinapant (0–1,000 nM) for 4, 8, 12, or 24 h, as indicated. GAPDH was used as a loading control.
Because pro-survival NF-jB signaling can interfere with cell death, we conducted a time course study to evaluate its activation post-Birinapant treatment. Data in Fig. 2f show that there is an initial increase in p-NFjB at 4 h with no change in total protein levels. However, at 8–12 h time points, a time-dependent decrease in both total and phos- phorylated NF-jB is observed. By 24 h, expression of both total and phosphorylated protein returns to untreated levels. Together, these results indicate the single agent efficacy of Smac mimetics in the SUM190 IBC cellular model.
Smac mimetics inhibit clonogenic growth capacity in combination with TRAIL and as single agents in SUM149 and SUM190 cells, respectively
To determine the effect of Smac mimetics on the growth characteristics of IBC cells, clonogenic growth potential was assessed. Data in Fig. 3a show that in TRAIL-sensitive SUM149 cells, Birinapant + TRAIL induces a significant reduction in clonogenic potential even at lower doses (40 % decrease in colony formation at 30 nM Birinapant +10 ng mL-1 TRAIL compared to 10 ng mL-1 TRAIL). Representative images show the decrease in the number of colonies formed with Birinapant + TRAIL versus TRAIL alone. Further, Birinapant was more potent than GT13402 in increasing TRAIL efficacy, with only 8 % colony for- mation efficiency at 100 nM Birinapant + 50 ng mL-1 TRAIL, compared to 32 % colony formation efficiency with 100 nM GT13402 + 50 ng mL-1 TRAIL (Fig. 3b).
Similar to our results from the trypan blue exclusion assay in Fig. 1, neither Birinapant nor GT13402 caused any change in SUM149 colony formation compared to untreated cells as single agents (data not shown).Unlike in SUM149 cells, both Smac mimetics were simi- larly effective at inhibiting the clonogenic growth potential of TRAIL-resistant SUM190 as single agents (Fig. 3c). Repre- sentative images show a decrease in SUM190 colony formation following treatment with Birinapant and GT13402.
Birinapant inhibits AIG potential
Anchorage-independent colony formation in soft agar has been widely used as a surrogate in vitro model to assess cancer cell tumorigenic potential and is considered to be a reasonably good predictor of in vivo activity [44, 45]. Data in Fig. 4a reveal that Birinapant + TRAIL (black bar) causes a 55 % reduction in the number of colonies formed, while TRAIL alone (white bar) causes a 20 % decrease relative to untreated. Representative images of colonies formed in soft agar in the right panel of Fig. 4a show similar sized colonies in all treatment groups, but a significant reduction in colony number of the Birinapant + TRAIL combination. Analysis of AIG in SUM190 cells, wherein we observed single agent efficacy of Birinapant, reveal a dose-dependent decrease in AIG following treatment with Birinapant (100–10,000 nM) (Fig. 4b). Data show a sig- nificant *50 % decrease in the number of colonies formed with 100 nM Birinapant treatment relative to untreated (Fig. 4b). Representative images of colonies formed in soft agar are shown in the right panel of Fig. 4b with no observable change in colony size, but a noticeable reduction in colony number with increasing dose of Birinapant.
Smac mimetic efficacy in SUM149 and SUM190 cells is TNF-a-independent and caspase-dependent
Studies were then conducted to evaluate whether TNF-a influences Smac mimetic efficacy in IBC cellular models. Since Smac mimetics, in addition to binding IAPs, have been shown to induce TNF-a production in sensitive cells, we measured TNF-a levels in the conditioned media of Birinapant-treated SUM149 and SUM190 cells by ELISA. Data in Fig. 5a reveal insignificant TNF-a secretion in SUM149. However, SUM190 cells, which are sensitive to Smac mimetics as single agents, produce autocrine TNF-a in a dose-dependent fashion in response to Birinapant treatment, although it should be noted that the levels were in the low picogram mL-1 range. Addition of exogenous TNF-a (50 ng mL-1) in combination with Birinapant in SUM190 cells had no significant additive effect on cell death compared to Birinapant alone as measured by trypan blue exclusion (Fig. 5b) and assessment of clonogenic growth capacity (Fig. 5c).
Next, the effect of TNF-a receptor (TNFR1) knockdown was evaluated in SUM190 cells using TNFR1-targeting siRNA and control scrambled siRNA at 24 h. Birinapant- induced cell death was not reversed (Fig. 6a) by specific TNFR1 downregulation (Fig. 6b). Furthermore, addition of a TNF-a neutralizing antibody (10 lg mL-1) as in a method described previously [46] to SUM149 (Fig. 7a, b) or SUM190 (Fig. 7c, d) cells did not reverse the decrease in viability or colony formation efficacy of Birinapant in combination or as a single agent.
In contrast to the TNF-a studies above, the apoptosis- inducing effect of Birinapant + TRAIL in SUM149 is potently reversed in the presence of pan-caspase inhibitor Q-VD-OPh, as evidenced by trypan blue exclusion and clonogenic growth assays (Fig. 7a, b). Similar reversal Birinapant or GT13402 (0–1,000 nM). Bars represent mean ± SEM colonies formed/cells plated as a percentage of the untreated sample. Representative images are shown on the right. c Clonogenic growth assay in SUM190 cells treated with Birinapant, GT13402 (0–10,000 nM) or TRAIL (0–100 mL-1). Bars represent mean ± – SEM colonies formed/cells plated as a percentage of the untreated sample (*P \ 0.05, #P \ 0.005; all comparisons made to the untreated sample). Representative images are shown on the right using Q-VD-OPh is observed in Birinapant-treated SUM190 cells (Fig. 7c, d). Together, these results reveal a TNF-a-independent but IAP- and caspase-dependent mechanism of action of Birinapant +TRAIL in SUM149 cells and Birinapant as a single agent in SUM190 cells.
Fig. 3 Smac mimetics inhibit clonogenic growth potential in com- bination with TRAIL in SUM149 cells and as single agents in SUM190 cells. a Clonogenic growth assay in SUM149 cells treated with TRAIL (10 ng mL-1) alone or in combination with Birinapant (0–1,000 nM). Bars represent mean ± SEM colonies formed/cells plated as a percentage of the untreated sample. Representative images are shown on the right. b Clonogenic growth assay in SUM149 cells treated with TRAIL (50 ng mL-1) alone or in combination.
Discussion
We report herein for the first time, IAP binding affinity of Birinapant, a bivalent Smac mimetic, and its efficacy in inducing apoptosis in TRAIL-sensitive and TRAIL-resis- tant IBC cellular models with differential XIAP expression. In the TRAIL-sensitive basal-type SUM149 cell line, the Smac mimetic Birinapant was not effective as a single agent, but significantly enhanced TRAIL potency in a dose- dependent manner. In contrast, in the TRAIL-resistant, ErbB2-overexpressing SUM190 cell line, Birinapant sig- nificantly decreased cell viability and clonogenic growth potential as a single agent. In addition, the effect of Biri- napant on these cellular models was further characterized using AIG as a predictor of in vivo behavior. The ability of cells in vitro to grow in the absence of anchorage has been shown to correlate directly with the ability of those cells to cause tumors in multiple mouse models [44, 47]. Thus, the inhibition of AIG seen in Birinapant-treated SUM190 cells and Birinapant + TRAIL-treated SUM149 cells highlights Birinapant’s ability to target tumorigenic cells and further supports Birinapant’s potential for efficacy in vivo.
IBC is a particularly aggressive subset of breast cancer that is characterized by rapid progression, local and distant metastasis, younger age of onset, and less than 5 % sur- vival rate beyond 5 years when treated with surgery or radiation therapy [1]. Apoptotic dysregulation has been observed in IBC cells and in patient tumors [2–4, 8, 9, 12, 13]. We have previously generated isogenic IBC cell models that are derived from SUM149 and SUM190 cell lines, in which therapeutic resistance to the dual ErbB1/2 tyrosine kinase inhibitor lapatinib is exhibited [2–4]. One of the dominant features in the resistant cell lines was overexpression of XIAP, which correlated directly with therapeutic resistance. We also reported that XIAP over- expression was not due to increased mRNA or protein stability, but rather due to increased IRES-mediated cap- independent translation of XIAP mRNA in response to lapatinib treatment [4]. The resistant cell lines also exhib- ited cross-resistance to other agents like TRAIL [5]. Thus, it is of particular interest to identify inhibitors of XIAP, as well as other members of the IAP family, in order to pre- vent or reverse acquired therapeutic resistance in IBC. In doing so, it may be possible to treat IBC with an IAP inhibitor alone, or to sensitize these tumors to other drugs as part of a combination regimen. One way to inhibit the IAP family of proteins is through the use of Smac mi- metics, which act in a manner similar to Smac/DIABLO, a known effector of IAP degradation in apoptosis initiation. In the present study, we characterized parental SUM149 and SUM190 IBC cell lines and their isogenic derivatives with differential XIAP expression (SUM149 wtXIAP, SUM190 shXIAP) for Smac mimetic sensitivity. These multiple lines were treated with Birinapant (pan-IAP antagonist with high affinity for cIAP1/2 and XIAP) and GT13402, a Smac mimetic with lower affinity for XIAP. The differential binding of these two new Smac mimetics provides a small molecule approach to probe the impact of XIAP inhibition on Smac mimetic treatment in IBC. Both Birinapant and GT13402 caused degradation of cIAP1 and cIAP2 but had no effect on XIAP levels. In contrast, it has been recently reported that bivalent Smac mimetic BV6 degraded both cIAP1 and XIAP but not cIAP2 in glio- blastoma cells, revealing differences in mechanism of action in different cell models [35]. In general, Smac mimetic compounds are more efficacious in enhancing potency of other chemotherapeutic agents [21]. In SUM149 cells, a similar phenomenon was observed as the cells were insensitive to Birinapant treatment alone, but the addition of Birinapant increased TRAIL efficacy.
Fig. 4 Birinapant inhibits anchorage-independent growth potential in combination with TRAIL in SUM149 cells and as a single agent in SUM190 cells. a Anchorage-independent growth assay in SUM149 cells treated with TRAIL (50 ng mL-1), Birinapant (1,000 nM), or the combination. Bars represent mean ± SEM colonies formed in soft agar as a percentage of the untreated sample (#P \ 0.005; comparisons made to TRAIL alone). Representative images are shown on the right. b Anchorage-independent growth assay of SUM190 cells treated with Birinapant (0–10,000 nM). Bars represent mean ± SEM colonies formed in soft agar as a percentage of the untreated sample (#P \ 0.005; all comparisons made to the untreated sample). Representative images are shown on the right.
A significant finding was the single agent efficacy of Bi- rinapant in the SUM190 cell line, which does not express death receptor DR4 and is insensitive to TRAIL treatment. Interestingly, previous studies have shown that Smac mi- metics in certain cancer models can cause paradoxical acti- vation of NF-jB by autocrine TNF-a production and that the efficacy of Smac mimetics can be blunted by this activation of pro-survival NF-jB signaling [27, 28, 36, 48–50], par- ticularly in an in vivo setting. However, in the SUM190 IBC cell model, it appears that Birinapant efficacy may not be affected by NF-jB activation since we observed transient changes, with slight activation and downregulation at earlier time points and activated and total NF-jB returning to basal levels by 24 h. Furthermore, this is supported by our obser- vations that Birinapant efficacy is not TNF-a-dependent. SUM190 cells showed only a modest dose-dependent increase in TNF-a levels at picogram mL-1 quantities in Birinapant-treated culture supernatants. This is interesting since pro-apoptotic activity of other Smac mimetics is often associated with significant autocrine production of TNF-a and interaction with death receptors [28, 36, 49]. A recent
Fig. 6 Inhibition of TNF-a signaling through TNFR1 knockdown does not inhibit Birinapant-mediated cell death in SUM190 cells. a Viability as measured by trypan blue exclusion assay of SUM190 cells with TNFR1 knockdown via siRNA or control siRNA treated with Birinapant (0–1,000 nM). Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 1–4). b Western immunoblot analysis of TNFR1 in control SUM190 cells (c) and those transfected with TNFR1-targeting siRNA or scrambled siRNA (scr). GAPDH was used as a loading control.
Birinapant + TRAIL treated SUM149 cells, where the addition of a neutralizing TNF-a antibody failed to inhibit cell death. Furthermore, addition of exogenous TNF-a did not enhance cell death in SUM190 after treatment with increasing doses of Birinapant, supporting TNF-a-indepen- dent efficacy of Birinapant in the IBC cells.
The present study shows Birinapant efficacy corresponded with significant cIAP1/2 degradation, apoptotic parameters, and a reversal of Birinapant-induced cell death after addition of a caspase inhibitor. A recent study has shown a monovalent Smac mimetic (LBW242) similarly induces cell death in a TNF-a-independent, caspase-dependent manner in neuroblastoma cell lines [51]. In contrast to Birinapant, LBW242 did not cause significant degradation of cIAP1/2. Further- more, the IC50 values for LBW242 as a single agent in the neuroblastoma cells lines were in the 50 lM concentration range, causing significant toxicity in matched normal cells. Birinapant, however, is efficacious at lower nanomolar con- centrations tested herein and previously reported [29]. Toge- ther, these results indicate that the mechanism of action of Birinapant in the IBC cell models tested is predominantly IAP-dependent, caspase-mediated apoptosis.
Fig. 5 SUM190 cells produce low levels of autocrine TNF-a after treatment with Birinapant, but exogenous TNF-a does not sensitize SUM190 cells to Birinapant. a TNF-a production as determined by ELISA in conditioned media from SUM149 and SUM190 cells treated with Birinapant (0–1,000 nM). Bars represent mean ± SEM (n = 3). b Viability as determined by trypan blue exclusion assay of SUM190 cells treated with Birinapant (0–1,000 nM) alone or in combination with TNF-a (50 ng mL-1). Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 2–4). c, Clonogenic growth assay of SUM190 cells treated with Birinapant (B; 0–1,000 nM) alone or in combination with TNF-a (50 ng mL-1). Bars represent mean ± SEM colonies formed/cells plated as a percentage of the untreated sample study in pancreatic cells with another Smac mimetic reported that inhibition of TNF-a by a neutralizing antibody reduced activation of caspases [50]. However, in this study, addition of a neutralizing TNF-a antibody or silencing of TNFR1 using siRNA failed to rescue SUM190 cells from Birinapant- induced cell death. A similar phenomenon was observed in
Currently, there is no standard IBC-specific treatment plan for patients with advanced disease. Birinapant is currently in a Phase 1a dose-escalation study in patients with refractory solid tumors and lymphoma [29]. By demonstrating that Birinapant primes IBC cells for death in an IAP-dependent manner, this study strengthens the fea- sibility of developing Birinapant for IBC therapy.
Fig. 7 Birinapant acts in a TNF-a-independent, caspase-dependent manner to kill SUM190 cells as a single agent and sensitize SUM149 cells to TRAIL. a Viability as determined by trypan blue exclusion assay of SUM149 cells treated with TRAIL (50 ng mL-1) in combi- nation with Birinapant (0–1,000 nM) with or without a TNF-a neutralizing antibody (10 lg mL-1) or pan-caspase inhibitor Q-VD- OPh (20 lM). Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 2–4, **P \ 0.01; all comparisons made between Birinapant + TRAIL and Birinapant + TRAIL + TNF-a Ab or Q-VD-OPh at the same concentration of Birinapant). b Clonogenic growth assay of SUM149 cells treated with TRAIL (50 ng mL-1) in combination with Birinapant (1,000 nM) with or without TNF-a Ab (10 lg mL-1) or pan-caspase inhibitor Q-VD-OPh (20 lM). Bars represent mean ± SEM colonies formed/cells plated as a percentage of the untreated sample. (#P \ 0.005; all comparisons made between Birinapant + TRAIL and Birinapant + TRAIL + TNF-a Ab or Q-VD-OPh at the same concentration of Birinapant). c Viability as determined by trypan blue exclusion assay of SUM190 cells treated with Birinapant (0–1,000 nM) in the presence of a TNF-a neutralizing antibody (10 lg mL-1) or the pan-caspase inhibitor Q-VD-OPh (20 lM). Data represent mean ± SEM viable cells taken as a percentage of total cells (n = 2–4, *P \ 0.05, **P \ 0.01; all com- parisons made between untreated and Q-VD-OPh-treated at the same concentration of Birinapant). d, Clonogenic growth assay of SUM190 cells treated with Birinapant (1,000 nM) in the presence of a TNF-a neutralizing antibody (10 lg mL-1) or the pan-caspase inhibitor Q-VD-OPh (20 lM). Bars represent mean ± SEM colonies formed/ cells plated as a percentage of the untreated sample. (*P \ 0.05; all comparisons made between TNF- a neutralizing antibody or Q-VD- OPh-treated at the same concentration of Birinapant).
Acknowledgments
The authors thank Amy Aldrich, Jess Hendin, and Katherine Aird for technical support. Grant support was received from American Cancer Society-RSG-08-290-01-CCE (GRD) and Duke University Chancellor’s Scholarship (JLA).
Conflict of interest GRD has previously received a philanthropic gift for breast cancer research from TetraLogic Pharmaceuticals.
Ethical standards This manuscript complies with the current laws of the United States of America.
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