TRAIL-induced apoptosis is preferentially mediated via …clincancerres.aacrjournals.org/content/clincanres/early/...phone: +49 69 67866557 Fax: +49 69 6786659157 Email: [email protected]

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research

TRAIL-induced apoptosis is preferentially mediated

via TRAIL receptor 1 in pancreatic carcinoma cells

and profoundly enhanced by XIAP inhibitors

1Dominic Stadel, 2Andrea Mohr 1Caroline Ref, 3Marion MacFarlane, 4Shaoxia Zhou, 5Robin Humphreys, 4Max Bachem, 3Gerry Cohen, 6Peter Möller, 2Ralf M. Zwacka 1Klaus-Michael

Debatin, 1,7Simone Fulda

1University Children’s Hospital, Ulm University, Ulm, Germany 2National University of Ireland, Galway, National Centre for Biomedical Engineering

Science, Galway, Ireland 3MRC Toxicology Unit, University of Leicester, Leicester, LE1 9HN, UK

4Department of Clinical Chemistry, Robert-Koch-Strasse 8, Ulm, Germany 5Oncology Research Department, Human Genome Sciences, Inc., Rockville, MD 20850, USA

6Institute of Pathology, Ulm University, Ulm, Germany 7 Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt,

Frankfurt, Germany

Running title: TRAIL-R1/-R2 signaling in pancreatic cancer Key words: TRAIL, apoptosis, XIAP, pancreatic cancer To whom correspondence and reprint requests should be addressed: Prof. Dr. Simone Fulda Institute for Experimental Cancer Research in Pediatrics Goethe-University Frankfurt Komturstr. 3a 60528 Frankfurt Germany phone: +49 69 67866557 Fax: +49 69 6786659157 Email: [email protected]

Published OnlineFirst on October 12, 2010 as 10.1158/1078-0432.CCR-10-0985

Research. on July 18, 2018. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 12, 2010; DOI: 10.1158/1078-0432.CCR-10-0985

http://clincancerres.aacrjournals.org/

TRAIL-R1/-R2 signaling in pancreatic cancer


2

Translational Relevance

Agonistic antibodies to the pro-apoptotic TRAIL receptors TRAIL-R1 (Mapatumumab) and

TRAIL-R2 (Lexatumumab) are currently under evaluation in early clinical trials in various

cancers including pancreatic cancer. However, it is not known at present which of the two

pro-apoptotic TRAIL receptors is better suited as a therapeutic target in pancreatic carcinoma.

The present study provides the first evidence that the majority of pancreatic carcinoma cell

lines and also primary cultured pancreatic carcinoma cells are more susceptible to

Mapatumumab compared to Lexatumumab especially in combination with XIAP inhibitors,

while Lexatumumab requires crosslinking for maximal activity, which may occur in vivo.

This preclinical evaluation of a rational combination of two novel classes of apoptosis-

targeting drugs, i.e. TRAIL receptor antibodies and XIAP inhibitors, in preclinical in vitro and

in vivo models of pancreatic cancer provides the molecular basis for the design of future

clinical studies and thus has important clinical implications.






3

Abstract

Purpose: We previously reported that small molecule XIAP inhibitors synergize with soluble

TRAIL to trigger apoptosis in pancreatic carcinoma cells. Since cancers may preferentially

signal via one of the two agonistic TRAIL receptors, we investigated these receptors as a

therapeutic target in pancreatic cancer in the present study.

Experimental Design: We examined TRAIL receptor expression and cytotoxicity of specific

monoclonal antibodies to TRAIL-R1 (HGS-ETR1, Mapatumumab) or TRAIL-R2 (HGS-

ETR2, Lexatumumab) and of TRAIL receptor selective mutants alone and in combination

with small molecule XIAP inhibitors in pancreatic cancer cell lines, primary specimens and in

a xenotransplant model in vivo.

Results: The majority of primary pancreatic carcinoma samples and all cell lines express one

or both agonistic TRAIL receptors. 9 of 13 cell lines are more sensitive to Mapatumumab-

induced apoptosis, while Lexatumumab requires crosslinking for maximal activity. Similarly,

TRAIL-R1 selective mutants display higher cytotoxicity than TRAIL-R2 selective mutants.

Small molecule XIAP inhibitors preferentially act in concert with Mapatumumab to trigger

caspase activation, caspase-dependent apoptosis and to suppress clonogenic survival. Also,

primary cultured pancreatic carcinoma cells are more susceptible to Mapatumumab than

Lexatumumab, which is significantly enhanced by a XIAP inhibitor. Importantly, combined

treatment with Mapatumumab and a XIAP inhibitor cooperates to suppress tumor growth in

vivo.

Conclusions: Mapatumumab exerts antitumor activity especially in combination with XIAP

inhibitors against most pancreatic carcinoma cell lines, while Lexatumumab requires

crosslinking for optimal cytotoxity. These findings have important implications for the design

of TRAIL-based protocols for pancreatic cancer.






4

Introduction

Pancreatic cancer is one of the leading causes of cancer deaths in the Western World (1).

Resistance of pancreatic cancer to even aggressive treatment regimens presents a major

challenge in oncology (2). Since evasion of apoptosis, the cell’s intrinsic cell death program,

contributes to treatment failure in pancreatic cancer (3, 4), current attempts to improve the

survival of pancreatic cancer patients will have to include strategies that target apoptosis

resistance.

Apoptosis pathways may be initiated through death receptors or mitochondria resulting in

caspase activation (5). Ligation of death receptors such as TNF-related apoptosis-inducing

ligand (TRAIL) receptors by their cognate ligands results in caspase-8 activation, which

induces direct cleavage of downstream effector caspases such as caspase-3 (6). The

mitochondrial pathway is engaged by the release of apoptogenic factors from mitochondria

into the cytosol, i.e. cytochrome c or second mitochondria-derived activator of caspase

(Smac)/direct IAP binding protein with low pI (DIABLO) (7). Cytochrome c triggers caspase-

3 activation via formation of the multimeric apoptosome complex, while Smac/DIABLO

promotes apoptosis by neutralizing ´Inhibitor of Apoptosis´ (IAP) proteins (7).

The concept of triggering TRAIL receptors on the cell surface to elicit apoptosis in cancer

cells is especially relevant for cancer therapy, since death receptors are directly linked to the

cell death program (6). To this end, TRAIL is considered as a prime candidate for clinical

application, because it has been reported to induce apoptosis in a panel of cancer cells without

limiting toxicity to normal human cells (8). However, many human cancers including

pancreatic carcinoma proved to be TRAIL resistant, e.g. because of high levels of IAP

proteins such as X-linked inhibitor of apoptosis (XIAP) (9, 10). XIAP prevents apoptosis at

the effector phase by binding to and inhibiting activated caspase-3 and -9 (11, 12). Since






5

XIAP blocks apoptosis at the core of the apoptotic machinery, therapeutic modulation of

XIAP can tackle a key control point in apoptosis resistance (11, 12).

Although TRAIL signals to apoptosis via either of the apoptosis-inducing TRAIL receptors

TRAIL-R1 and TRAIL-2, it has initially been assumed that it is in particular TRAIL-R2 that

plays a dominant role in initiating apoptosis (13). The higher expression of TRAIL-R2 on

many cancer cell lines has been put forward as an argument to support this concept, although

a clear relationship between receptor expression levels and the response to either TRAIL-R1

or TRAIL-2 activating compounds has not been established (13). More recently, the concept

that it is predominately TRAIL-R2 that mediates TRAIL-induced apoptosis has also been

challenged by data showing that some cancers, for example chronic lymphocytic leukemia

(CLL), predominately signal to cell death via TRAIL-R1 (14, 15).

We previously reported that inhibition of XIAP profoundly enhances TRAIL-induced

apoptosis in pancreatic carcinoma in vitro and in vivo (16-18). Besides soluble TRAIL,

specific TRAIL receptor antibodies have been developed for clinical application, which

demonstrate promising activities in early clinical trials (13, 19-21). TRAIL-R1 monoclonal

antibodies have already been administered to pancreatic carcinoma patients in a phase I

clinical trial (22). However, the question which of the two agonistic TRAIL receptors is in

fact better suited as a therapeutic target in pancreatic cancer has not yet been answered. So

far, no parameters have been identified that can accurately predict upfront whether a given

tumor responds better to TRAIL-R1 versus –R2 stimulation. This highlights the need to

evaluate the efficacy of TRAIL-R1 and –R2 specific antibodies in preclinical models of

pancreatic cancers. Therefore, we investigated the effects of fully human monoclonal

antibodies that bind specifically to either TRAIL-R1 (Mapatumumab) or TRAIL-R2

(Lexatumumab) in pancreatic carcinoma in the present study.






6






7

Materials and Methods

Cell culture and reagents

Pancreatic carcinoma cells were cultured in DMEM, DMEM/F12 or RPMI1640 (Life

Technologies, Inc., Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS)

(Biochrom, Berlin, Germany), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin

(Biochrom) and 25 mM HEPES (Biochrom) as described (17). A culture was established from

primary pancreatic carcinoma cells (ULA) derived from a peritoneal metastasis of a 71 year

old female patient with pancreatic adenocarcinoma and subsequently used at low passage

numbers. Genotypic characterization showed homozygous deletion of p16 and Smad4, single

deletion of p53, loss of heterozygosity of LKB1, wildtype status for PRSS1 and normal

expression of MLH1. TRAIL was purchased from R&D Systems, Inc. (Wiesbaden, Germany)

and TRAIL receptor specific mutants were described previously (14). The fully human

agonist monoclonal antibodies against TRAIL-R1 and TRAIL-R2, Mapatumumab and

Lexatumumab, respectively, were kind gifts from Human Genome Sciences (13). XIAP

inhibitor 1, XIAP inhibitor 2 and control compound correspond to compounds 2, 11 and 15,

respectively, described by Oost et al. (23). XIAP inhibitors 3 and 4 were described by Chao et

al. (24) and were kindly provided by Idun Pharmaceuticals now Pfizer, Inc. (Groton, CN).

XIAP inhibitors are capped tripeptides consisting of unnatural amino acids that were designed

on the basis of the NMR structure of a Smac peptide bound to the BIR3 domain of XIAP and

bind to XIAP BIR3 with high nanomolar affinities (23). The broad range caspase inhibitor

zVAD.fmk was purchased from Bachem (Heidelberg, Germany). All chemicals were

purchased from Sigma unless indicated otherwise.






8

Determination of apoptosis, cell viability and clonogenic survival

Apoptosis was determined by fluorescence-activated cell-sorting analysis (FACScan, BD

Biosciences, Heidelberg, Germany) of DNA fragmentation of propidium iodide-stained nuclei

(25). Cell viability was assessed by MTT assay according to the manufacturer´s instructions

(Roche Diagnostics, Mannheim, Germany). For clonogenic assay, cells were seeded as single

cells (0.02 x 105 cells/cm2) in 6-well plates for 24h, treated with TRAIL receptor antibodies

and/or XIAP inhibitor for 24h (PancTu1) or 3h (PaTuII) before medium was exchanged,

colonies were stained after an additional 10 days with crystal violet solution (0.75% crystal

violet, 50% ethanol, 0.25% NaCl and 1.57% formaldehyde).

Western blot analysis

Western blot analysis was performed as described (17) using the following antibodies: mouse

anti-caspase-8 (ApoTech Corporation, Epalinges, Switzerland), rabbit anti-caspase-3 (Cell

Signaling, Beverly, MA), rabbit anti-caspase-9 and mouse anti-XIAP from BD Biosciences

(Heidelberg, Germany), rabbit anti-cIAP2 (Epitomics, Burlingame, CA), goat anti-cIAP1 and

rabbit anti-survivin (R&D Systems, Inc.) or mouse anti-β-actin (Sigma) followed by goat-

anti-mouse IgG or goat-anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz

Biotechnology, Santa Cruz, CA). Enhanced chemiluminescence was used for detection

(Amersham Bioscience, Freiburg, Germany).

TRAIL receptor surface staining

To determine surface expression of TRAIL receptor cells were incubated with mouse anti-

human TRAIL-R1 to –R4 monoclonal antibodies (all from ApoTech Corporation, Epalinges,

Switzerland) for 30 min at 4°C, washed in PBS containing 1% FCS, incubated with rabbit

anti-mouse-F(ab´)2IgG/Biotin (BD Biosciences) for 20 min at 4°C in the dark, washed in PBS






9

containing 1% FCS, incubated with streptavidin-PE (BD Biosciences) for 20 min at 4°C in

the dark and analyzed by flow cytometry.






10

Immunohistochemistry

Immunohistochemistry of TRAIL receptors was performed on 24 pancreatic ductal

adenocarcinomas and 4 normal pancreata as previously described (26). Briefly, 2 µm-thick

cryosections were immediately fixed in ice-cold acetone for 10 min, air- dried and incubated

for 1 h with mouse monoclonal antibodies to TRAIL-R1 (clone HS101; IgG1 isotype),

TRAIL-R2 (clone HS201; IgG1 isotype), TRAIL-R3 (clone HS301, IgG1 isotype) or TRAIL-

R4 (clone HS401; IgG1 isotype), respectively, in a dilution of 1:100 (Alexis, San Diego, CA).

Bound primary antibody was detected via the REAL EnVision Detection System

Peroxidase/DAB+ (K5007; Dako, Glostrup, Denmark) followed by hematoxylin

counterstaining. TRAIL-R expression using the above monoclonal antibodies yielded

identical results in normal pancreata as obtained in a previous study by different antibodies to

these receptors (26). Negative controls were performed by omitting the first antibody and

yielded negative stainings (Fig. 1A, a). Results of immunohistochemistry were scored as

“negative”, n; “weakly positive”, wp; “positive”, p; “strongly positive”, sp. In cases of

staining heterogeneity within the target cell population two modalities were allowed, e.g.

“p/n“, and not further quantified.

Chorioallantoic membrane assay

Chorionallantoic membrane (CAM) assay was done as described previously (16). Briefly,

1x106 tumor cells were resuspended in 10 µl serum-free medium and 10 µl Matrigel Matrix

(BD Biosciences) and implanted on fertilized chicken eggs on day 8 of incubation. Tumors

were topically treated with 0.25 µg Mapatumumab or Lexatumumab diluted in 15 µl serum-

free medium with or without 10 µM XIAP inhibitor daily for 3 days, sampled with

surrounding CAM 4 days after seeding, fixed in 4% paraformaldehyde, paraffin embedded,

cut in 5 µm sections and hematoxylin/eosin stained. Tumor area was measured in a






11

representative picture of each tumor and the percentage of cellular area of the whole tumor

area was calculated using OPTIMAS 6.5.1 (Media Cybernetics, Bethesda, MD). In detail,

whole tumor area of each picture was marked as region of interest, including tumor cells,

matrigel and CAM tissue. Color threshold was set for viable hematoxylin/eosin stained tumor

cells and the threshold reaching area containing viable tumor cells was calculated and

expressed as percentage of region of interest.

Statistical analysis

Statistical significance was assessed by two-sided Student's t-test using Microsoft® Excel®

(Microsoft Deutschland GmbH, Unterschleißheim, Germany). Interaction between XIAP

inhibitors and TRAIL receptor antibodies was analyzed by the Combination index (CI)

method based on that described by Chou (27) using CalcuSyn software (Biosoft, Cambridge,

UK). Combination index (CI) <0.9 indicates synergism, 0.9-1.1 additivity and >1.1

antagonism.






12

Results

Recently, we reported that small molecule XIAP inhibitors synergize with TRAIL to trigger

apoptosis in pancreatic carcinoma cells in vitro and in vivo (16). Since it is not known at

present which of the two agonistic TRAIL receptors is superior as a therapeutic target in

pancreatic carcinoma, we evaluated agonistic TRAIL-R1 and -R2 specific antibodies alone

and in combination with XIAP inhibitors in the present study.

Expression of TRAIL receptors in pancreatic carcinoma

First, we explored the expression status of TRAIL receptors in human primary pancreatic

ductal adenocarcinoma samples and in normal pancreatic tissue by immunohistochemistry.

Ducts of normal, non-inflammed pancreata were consistently negative for all TRAIL

receptors (not shown), confirming our previously published data (26). Expression data of

pancreatic carcinomas are listed in Tab. 1. Of 24 carcinomas 12 were at least in part induced

for TRAIL-R1 expression compared to normal pancreatic ducts, seven of which were entirely

TRAIL-R1 positive (Fig. 1A, b). TRAIL-R2 was expressed in 18/24 carcinomas, 12 of which

were entirely TRAIL-R2 positive including one with strong TRAIL-R2 expression throughout

(Fig. 1A, c). TRAIL-R3 was expressed in 14/24 carcinomas, often only in subsets of

neoplastic cells and only once in a weak manner (Fig. 1A, e). TRAIL-R4 was most frequently

induced in pancreatic carcinomas compared to normal pancreatic tissue (22/24 samples) (Fig.

1A, f). Regarding the TRAIL-receptor expression profile, there was no entirely TRAIL-R1 to

-R4 positive nor an entirely TRAIL-R1 to -R4 negative case, however, four cases lacked both,

TRAIL-R1 and –R2 (Tab. 1). No obvious correlation of TRAIL receptor expression and grade

of differentiation was observed (Tab. 1).

Next, we examined cell surface expression of TRAIL receptors in a panel of pancreatic

carcinoma cell lines. All cell lines exhibited surface expression of the two agonistic TRAIL






13

receptors TRAIL-R1 and -R2 (Fig. 1B and (17)). TRAIL-R3 and TRAIL-R4 were expressed

at low or undetectable levels in most cell lines except PaTu8988t, T3M4, ASPC1 and PaTuII

that express considerable levels of TRAIL-R4 (Fig. 1B and (17).

XIAP inhibitor preferentially cooperates with Mapatumumab to reduce viability in

pancreatic carcinoma cells

To gain insight into the regulation of TRAIL-induced apoptosis via TRAIL-R1 and TRAIL-

R2 in pancreatic carcinoma cells, we analyzed the cytotoxicity of fully human monoclonal

antibodies specifically directed against TRAIL-R1 (Mapatumumab) and TRAIL-R2

(Lexatumumab). Interestingly, Mapatumumab was more potent to reduce cell viability than

Lexatumumab in the majority of pancreatic carcinoma cell lines (9 of 13 cell lines), while

four cell lines (MiaPaCa2, PaTu8988t, PaTu8988s, ASPC1) were more susceptible to

Lexatumumab (Fig. 2A).

Next, we assessed the effect of TRAIL receptor specific antibodies in combination with a

small molecule XIAP inhibitor that binds to the BIR3 domain of XIAP (23). Of note, the

XIAP inhibitor significantly enhanced loss of viability in combination with one of the

agonistic TRAIL receptor antibodies in all cell lines investigated (Fig. 2A). The majority of

pancreatic carcinoma cell lines (9 of 13 cell lines) were more susceptible to the combination

of Mapatumumab and the XIAP inhibitor compared to Lexatumumab (Fig. 2A). By

comparison, the four cell lines that were more responsive to treatment with Lexatumumab

alone (MiaPaCa2, PaTu8988t, PaTu8988s, ASPC1) were also more sensitive to the

combination of Lexatumumab plus XIAP inhibitor (Fig. 2A).

Moreover, we simultaneously treated cells with Mapatumumab and Lexatumumab to test

whether the concomitant stimulation of both agonistic TRAIL receptors results in additive,

synergistic or antagonistic cytotoxicity. The combined use of Mapatumumab and






14

Lexatumumab neither acted in concert to reduce viability nor antagonized each other, in the

presence or absence of the XIAP inhibitor (Fig. 2A). The only exception were MiaPaCa2

cells, where simultaneous treatment with Mapatumumab and Lexatumumab resulted in

enhanced reduction of cell viability compared to treatment with Mapatumumab and

Lexatumumab alone (Fig. 2A). Control experiments using a close structural analogue that

weakly binds to XIAP (23) showed no cooperative interaction with either of the TRAIL

receptor antibodies (Suppl. Fig. 1). Together, this set of experiments demonstrates that the

majority of pancreatic carcinoma cell lines are more susceptible to TRAIL-R1 than TRAIL-

R2 specific antibodies, either as single agents or in combination with a small molecule XIAP

inhibitor. For further studies, we selected PancTu1 and PaTuII pancreatic carcinoma cells as

prototype cell lines, which preferentially respond to Mapatumumab in combination with the

XIAP inhibitor.

Distinct XIAP inhibitors preferentially cooperate with Mapatumumab to induce

apoptosis in pancreatic carcinoma cells

To test the broader relevance of our findings, we extended our studies to additional,

structurally modified XIAP inhibitors, which all bind to the same surface groove of the BIR3

domain of XIAP (23, 24). Distinct XIAP inhibitors profoundly enhanced Mapatumumab-

induced loss of viability, while they displayed a minor cooperative interaction with

Lexatumumab (Fig. 2B, Suppl. Fig. 2). The simultaneous stimulation with Mapatumumab and

Lexatumumab resulted in a similar reduction of cell viability compared to stimulation with

Mapatumumab alone, either in the absence or in the presence of XIAP inhibitors (Fig. 2B,

Suppl. Fig. 2). Dose titration studies of Mapatumumab and XIAP inhibitor 3 revealed that the

interaction of these two agents was highly synergistic (Fig. 2C, Tab. 2).






15

To confirm that cells die by apoptotic cell death, we assessed DNA fragmentation as a

characteristic feature of apoptosis. Importantly, addition of XIAP inhibitors profoundly

increased Mapatumumab-induced apoptosis in a dose- and time-dependent manner (Fig. 3A,

3B and Suppl. Fig. 3). By comparison, no or only a slight augmentation of apoptosis was

observed, when the XIAP inhibitor was combined with Lexatumumab (Fig. 3A and 3B).

Furthermore, we performed colony assays to examine the long-term effects of the

combination treatment. The XIAP inhibitor preferentially cooperated with Mapatumumab to

suppress colony formation compared to Lexatumumab (Fig. 3C). The specificity of

Mapatumumab and Lexatumumab for TRAIL-R1 and TRAIL-R2, respectively, was

confirmed by RNAi-mediated knockdown (Suppl. Fig. 4). Together, this set of experiments

demonstrates that inhibition of XIAP preferentially sensitizes pancreatic carcinoma cells for

TRAIL-R1-mediated apoptosis, resulting in long-term suppression of clonogenic survival.

Preferential activity of TRAIL-R1 selective mutants against pancreatic carcinoma cells

To further explore the susceptibility of pancreatic carcinoma cells towards TRAIL-R1 versus

TRAIL-R2 stimulation, we used mutant forms of TRAIL that bind to TRAIL-R1 or TRAIL-

R2 with high specificity (14). In combination with the XIAP inhibitor, the TRAIL-R1

selective mutant was more potent than the TRAIL-R2 selective mutant to reduce viability of

PancTu1 and PaTuII cells (Fig. 4A). These results confirm the findings with TRAIL receptor

specific antibodies that pancreatic carcinoma cells are more susceptible to TRAIL-R1

triggering.

Crosslinking increases Mapatumumab’s activity when combined with XIAP inhibitor

Since TRAIL-R2 has previously been reported to require crosslinking for its full activity (28),

we next investigated whether a crosslinking agent augments the cytotoxicity of TRAIL






16

receptor antibodies. In the presence of the XIAP inhibitor, crosslinking of Lexatumumab

significantly increased its cytotoxicity, whereas crosslinking of Mapatumumab did not alter

its cytotoxicity (Fig. 4B). In the absence of the XIAP inhibitor however, the addition of a

crosslinker had no or a minor effect on the cytotoxicity of TRAIL receptor antibodies (Fig.

4B). These findings demonstrate that crosslinking enhances Lexatumumab-induced

cytotoxicity either alone or when XIAP is simultaneously neutralized.

XIAP inhibitor preferentially cooperates with Mapatumumab to trigger caspase

activation

To gain insight into the activation of the TRAIL signalling cascade upon triggering of

TRAIL-R1 or TRAIL-R2 and its modulation by XIAP inhibitors, we monitored cleavage of

caspases by Western blotting. In both PancTu1 and PaTuII cells, Mapatumumab alone was

more potent to induce cleavage of caspase-9 and -3 into active fragments compared to

Lexatumumab (Fig. 5A). Also, the XIAP inhibitor preferentially cooperated with

Mapatumumab to enhance caspase activation (Fig. 5A). Treatment with the XIAP inhibitor

alone did not initiate caspase cleavage (Fig. 5A), consistent with our findings that the

concentration of XIAP inhibitor used in these experiments is subtoxic and insufficient to

initiate apoptosis in the absence of an additional pro-apoptotic stimulus (Fig. 2 and 3). We

also assessed caspase activity by enzymatic caspase assays. Similarly, the XIAP inhibitor

preferentially acted in concert with Mapatumumab to increase caspase activity compared to

Lexatumumab (Fig. 5B, Suppl. Fig. 5). To test the requirement of caspase activity for

apoptosis induction we used the broad range caspase inhibitor zVAD.fmk. Addition of

zVAD.fmk almost completely rescued loss of viability upon the combination treatment with

Mapatumumab or Lexatumumab and XIAP inhibitor (Fig. 5C), demonstrating that loss of

viability occurred in a caspase-dependent manner.






17

No preferential activation of PI3K/Akt/mTOR or Raf/MEK/ERK survival pathways by

Mapatumumab or Lexatumumab

Since TRAIL has been reported to stimulate survival signalling such as the PI3K/Akt/mTOR

and Raf/MEK/ERK pathways besides the induction of apoptosis (29), we asked whether

TRAIL-R1 and TRAIL-R2 may differentially activate these survival cascades in pancreatic

carcinoma cells. To address this question we monitored the phosphorylation status of both

upstream and downstream components of the PI3K/Akt/mTOR pathway using Akt as a target

of PI3K, S6 ribosomal protein as a target of mTOR and ERK as a component of the

Raf/MEK/ERK pathway. Treatment with Mapatumumab or Lexatumumab did not increase

phosphorylation of Akt, S6 ribosomal protein or ERK compared to control cells treated with

solvent (Suppl. Fig. 6). This indicates that the reduced cytotoxicity of Lexatumumab over

Mapatumumab is not simply due to preferential activation of the PI3K/Akt/mTOR and/or

Raf/MEK/ERK pathways by Lexatumumab.

Mapatumumab shows higher activity than Lexatumumab against primary cultured

pancreatic carcinoma cells

In order to validate the results obtained in cell lines, we extended our studies to primary

cultured pancreatic carcinoma cells derived from a pancreatic adenocarcinoma specimen.

Primary cultured pancreatic carcinoma cells (ULA) express TRAIL-R1, -R2 and -R4 as well

as cIAP1, cIAP2, XIAP and survivin protein (Fig. 6A and 6B). At equimolar concentrations,

Mapatumumab was more potent than Lexatumumab to reduce cell viability of primary

cultured pancreatic carcinoma cells (Fig. 6C). Importantly, the addition of the XIAP inhibitor

further enhanced Mapatumumab-mediated loss of viability (Fig. 6C). These findings






18

demonstrate that Mapatumumab exerts higher cytotoxicity against primary cultured pancreatic

carcinoma cells compared to Lexatumumab alone or in combination with XIAP inhibitor.

Mapatumumab cooperates with XIAP inhibitor to suppress pancreatic carcinoma

growth in vivo

Finally, we extended our studies to an in vivo setting, using the CAM model as an established

in vivo tumor model that allows the assessment of antitumor activity in a three dimensional

setting (16). Mapatumumab together with XIAP inhibitor significantly reduced tumor growth

compared to untreated tumors (Fig. 6D). Also, the combined treatment with Lexatumumab

and the XIAP inhibitor exerted some antitumor activity compared to the control, although this

did not reach statistical significance (Fig. 6D). This demonstrates that although both

Mapatumumab and Lexatumumab can act in concert with XIAP inhibitor to suppress

pancreatic carcinoma growth in vivo Mapatumumab is significantly more potent than

Lexatumumab.






19

Discussion

TRAIL receptor agonists are currently evaluated in early clinical trials in a variety of tumors

including pancreatic cancers (13, 19-22, 30). There is mounting evidence that individual

cancers preferentially signal via one of the agonistic TRAIL receptors (14, 15). In pancreatic

carcinoma however, it has not yet been explored whether one of the two proapoptotic TRAIL

receptors is superior as a therapeutic target.

Here, we provide first evidence that the majority of pancreatic carcinoma cell lines and

also primary cultured pancreatic carcinoma cells are more sensitive to TRAIL-R1 over

TRAIL-R2 agonists, especially in combination with XIAP inhibitors, and that crosslinking is

required for maximal activity of the TRAIL-R2 antibody Lexatumumab. This conclusion is

supported by two distinct approaches to trigger one of the agonistic TRAIL receptors: Firstly,

fully human monoclonal antibodies that specifically bind with a 1000 times greater affinity to

either TRAIL-R1 or TRAIL-R2 (13) and secondly, TRAIL receptor-selective mutants (14).

Data obtained in a panel of pancreatic carcinoma cell lines underline the generality of the

results and experiments using an established culture of clinical tumor material confirm the

clinical relevance of the findings.

The molecular basis of the preferential signaling via one of the two agonistic TRAIL

receptors in cancer cells that express both receptors is currently not exactly known. While our

data demonstrate no differential activation of cell survival signaling, i.e. activation of the

PI3K/Akt/mTOR or Raf/MEK/ERK pathways, upon triggering of TRAIL-R1 or TRAIL-R2,

they reveal clear differences in the crosslinking requirements. Accordingly, crosslinking of

TRAIL-R2 profoundly enhances its cytotoxicity either alone or when XIAP is concomitantly

antagonized, while crosslinking of TRAIL-R1 has no or minimal additional effects. Thus, the

relatively low susceptibility of pancreatic cancer cells to TRAIL-R2 antibodies can be

overcome by increasing the crosslinking status of TRAIL-R2. Distinct crosslinking






20

requirements have previously been reported for TRAIL-R1 and TRAIL-R2. It is in particular

TRAIL-R2 that is described to require crosslinking to enhance its apoptosis inducing activity

(28, 31, 32) and that triggers apoptosis under certain conditions only when crosslinked (33).

In vivo, antibody crosslinking can occur via Fc receptors of immune effector cells (34) or via

the complement system (35). This may account for some antitumor activity of Lexatumumab

and XIAP inhibitor that we observed in our in vivo experiments, as an inflammatory reaction

to exogenous material of the CAM of chicken embryos has been reported (36) and Fc receptor

bearing cells have been described in the intra-embryonic mesenchyme of chicken embryos

(37). Hence, partial crosslinking of Lexatumumab by the host´s immune system might occur

in the CAM model in vivo. It will be interesting to investigate whether different TRAIL-R2

agonists that are under (pre)clinical evaluation differ in their ability to crosslink TRAIL-R2

and thus, may vary in their anticancer activity.

Furthermore, our findings reveal that the susceptibility of pancreatic carcinoma cells to

TRAIL-R1 or TRAIL-R2 ligation does not directly correlate with surface expression of the

respective TRAIL receptors. This observation is in line with previous studies showing that

membrane expression of agonistic TRAIL receptors does not directly link to the cell’s

susceptibility towards TRAIL-R1 or TRAIL-R2 stimulation (14, 15, 28, 31, 38). For example,

CLL cells were found to signal primarily via TRAIL-R1, although they express TRAIL-R1

and TRAIL-R2 at similar levels. Vice versa, lung, colon and breast carcinoma cell lines, all

with similar membrane levels of TRAIL-R1 and TRAIL-R2, displayed a higher sensitivity to

TRAIL-R2 selective mutants (31). By comparison, the preferential response of ovarian, colon

and renal cell carcinoma cell lines to TRAIL-R2 antibodies was associated with higher

surface expression levels of TRAIL-R2 (32, 39-41). Although a more widespread and higher

expression of TRAIL-R2 has been reported in a number of studies, no clear relationship

between TRAIL receptor levels and the response of cancer cells to targeting one or other of






21

the agonistic TRAIL receptors has been established (13). Thus, the relative contribution of

each of the agonistic TRAIL receptors to initiate apoptosis in cells that express both receptors

is not a simple consequence of surface expression levels and might be determined by

intracellular regulators of apoptosis. This highlights the importance of functional (pre)clinical

studies such as the present one to identify the TRAIL receptor subtype that may preferentially

or exquisitely transmit the apoptotic signal in a given type of cancer.

From the translational perspective of targeting TRAIL receptors in pancreatic cancer it is

important to note that neoexpression of all TRAIL receptors was found in malignant versus

non-malignant pancreatic carcinoma tissue, consistent with our previous findings showing the

absence of TRAIL receptors in normal, non-inflamed pancreata (26). A recent

immunohistochemical study similarly showed upregulation of TRAIL-R1 and TRAIL-R4 in

pancreatic carcinoma tissue compared to the normal pancreas (42). Together, these findings

suggest that the TRAIL receptor system may serve as therapeutic targets in pancreatic cancer.

Another important finding of this study is that simultaneous inhibition of XIAP enhanced

TRAIL-R1- or TRAIL-R2-induced apoptosis in a highly synergistic manner. This is

particularly relevant, since TRAIL receptor antibodies as monotherapy displayed limited

antitumor activity in the majority of the pancreatic cancer cell lines investigated, in line with

our previous results for soluble recombinant TRAIL (17). This indicates that combination

regimens to enhance the therapeutic potential of TRAIL receptor agonists are required to

ensure the success of TRAIL receptor agonists against pancreatic cancer. Previously, we

demonstrated that neutralizing XIAP either by RNA interference-mediated knockdown or by

small molecule inhibitors acted in concert with soluble recombinant TRAIL to induce

apoptosis in pancreatic cancer in vitro and in vivo (16-18). In addition, XIAP small molecule

antagonists that target the BIR2 domain of XIAP were reported to synergize with TRAIL in

pancreatic cancer (17, 43). Compared to these earlier reports that focus on soluble






22

recombinant TRAIL, the current study shows for the first time that the antitumor activity of

TRAIL receptor specific antibodies is profoundly enhanced by neutralizing XIAP in

pancreatic cancer cells. These findings support the concept that simultaneous targeting of

XIAP in combination with stimulation of the TRAIL pathway is a promising approach to

augment the antitumor activity of TRAIL receptor agonists against pancreatic cancer. They

also have important implications for the design of future clinical trials with TRAIL receptor

antibodies for the treatment of pancreatic cancer, since Mapatumumab and Lexatumumab are

already under clinical evaluation alone or in combination with chemotherapy (22). The

clinical relevance of our findings is supported by data obtained in primary cultured pancreatic

carcinoma cells that were established from a tumor specimen of a patient with pancreatic

adenocarcinoma. Although the analysis of primary material is so far restricted to one

specimen, the data provide a first proof-of-concept that the reported findings are not restricted

to established cell lines, but are also relevant for patients’ derived primary tumor cells

established in culture.

In conclusion, this preclinical evaluation of a rational combination of two novel classes of

apoptosis-targeting drugs, i.e. TRAIL receptor antibodies and XIAP inhibitors, in relevant

preclinical in vitro and in vivo models of pancreatic cancer provides the molecular basis for

the design of new combination therapies for the treatment of pancreatic cancer. This strategy

may help to overcome apoptosis resistance of pancreatic cancer, one of the cancers with the

worst prognosis.

Acknowledgements

We thank C. Hulford and M. Luzzio (Pfizer Inc., Groton, CN) for providing XIAP inhibitor,

A. Dittrich and E. Scheidhauer for excellent technical assistance and B. Welz for expert






23

secretarial work. This work has been partially supported by grants from the Deutsche

Forschungsgemeinschaft, the Deutsche Krebshilfe, the European Community (ApopTrain,

APO-SYS) and IAP6/18 (to S. F.).






24

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29

Figure legends

Figure 1. TRAIL receptor expression in pancreatic carcinoma

A, TRAIL receptor expression was analyzed by immunohistochemisty on frozen sections in

24 samples of pancreatic ductal adenocarinoma and 4 samples of normal pancreas. Examples

of a case of a moderately differentiated (case number 11; a-c) and a poorly differentiated (case

number 15; d-f) ductal pancreatic adenocarcinoma are presented. Case 11 shows weak

TRAIL-R1 expression in all tumor cells (b) and TRAIL-R2 expression at a clearly higher

level (c) (a is the negative control). Case 15 expresses TRAIL-R1 only weakly and in a

minority of neoplastic cells (d) and has clear-cut TRAIL-3 expression in a subset of neoplastic

cells (e) while featuring strong TRAIL-R4 expression (f). (Scale bar in a, valid for a-c,

corresponds to 240 µm; scale bar in d, valid for d-f, corresponds to 120 µm).

B, Surface expression of TRAIL receptors 1-4 on pancreatic carcinoma cell lines was

determined by flow cytometry (thin line: isotype control, thick line: anti-TRAIL receptor

antibodies). Fluorescence intensity (x-axis) is blotted against cell counts (y-axis). A

representative experiment of 3 independent experiments is shown.

Figure 2. XIAP inhibitors cooperate with TRAIL receptor antibodies to reduce viability

of pancreatic carcinoma cells.

A, Cells were treated for 48h with indicated concentrations of Mapatumumab, Lexatumumab

or both Mapatumumab and Lexatumumab and/or 10 μM XIAP inhibitor 2 or DMSO. Cell

viability was determined by MTT assay and is expressed as percentage of untreated controls.

B, PancTu1 (left panels) and PaTuII (right panels) cells were treated for 48h with indicated

concentrations of Mapatumumab (upper panels), Lexatumumab (middle panels) or

Mapatumumab and Lexatumumab (lower panels) and/or a subtoxic concentration (10 μM) of

XIAP inhibitor 3 or DMSO.






30

C, Cells were treated for 48h with indicated concentrations of Mapatumumab and indicated

concentrations of XIAP inhibitor 3. Cell viability was determined by MTT assay and is

expressed as percentage of untreated controls. Mean + SEM of three independent experiments

performed in triplicate are shown; #, P<0.05; *, P<0.01 comparing XIAP inhibitors to

solvent.

Figure 3. XIAP inhibitor preferentially cooperates with Mapatumumab to induce

apoptosis and to reduce colony formation.

PancTu1 (left panel) and PaTuII (right panel) cells were treated with Mapatumumab,

Lexatumumab or both Mapatumumab and Lexatumumab at indicated concentrations (A) or 3

μg/ml (B) and/or 10 μM XIAP inhibitor 3 or DMSO. Apoptosis was determined by FACS

analysis of DNA fragmentation of propidium iodide stained nuclei. In C, colony formation

after treatment with Mapatumumab or Lexatumumab at 3 μg/ml (left panel) or 1 μg/ml (right

panel) and/or 10 μM XIAP inhibitor 3 or DMSO was assessed by crystal violet staining. A

representative experiment of three independent experiments is shown.

Figure 4. XIAP inhibitor preferentially cooperates with TRAIL-R1 selective mutant to

induce apoptosis whereas crosslinking of TRAIL-R2 potentiates apoptosis.

PancTu1 (left panels) and PaTuII (right panels) cells were treated for 48h with wildtype

soluble TRAIL, TRAIL-R1 selective mutant (R1-5) or TRAIL-R2 selective mutant (R2-6) or

with Mapatumumab and Lexatumumab and/or 10 μM XIAP inhibitor 3 or DMSO (A). In B,

Mapatumumab and Lexatumumab were used in the absence or presence of crosslinker. Cell

viability was determined by MTT assay and is expressed as percentage of untreated controls.

Mean + SEM of three independent experiments performed in triplicate are shown.






31

Figure 5. XIAP inhibitor enhances TRAIL-induced activation of caspases.

A, PancTu1 (upper panel) and PaTuII (lower panel) cells were treated for indicated times with

3 μg/ml Mapatumumab or Lexatumumab and/or 10 μM XIAP inhibitor 3 or DMSO. Caspase

activation was determined by Western blotting. Arrows indicate caspase cleavage fragments.

For pro-caspase-8, short and long exposures of the blots are presented. A representative

experiment of three independent experiments is shown.

B, PancTu1 (left panel) and PaTuII (right panel) cells were treated for indicated times with 3

μg/ml (PancTu1) or 1 μg/ml (PaTuII) Mapatumumab or Lexatumumab and/or 10 μM XIAP

inhibitor 3 or DMSO. Caspase activity was assessed by caspase assay.

C, PancTu1 (left panel) and PaTuII (right panel) cells were treated for 48h with 3 μg/ml

(PancTu1) or 1 μg/ml (PaTuII) Mapatumumab or Lexatumumab and/or 10 μM XIAP

inhibitor 3 or DMSO in the presence or absence of 25 µM zVAD.fmk. Cell viability was

determined by MTT assay and is expressed as percentage of untreated controls. Mean + SEM

of three independent experiments performed in triplicate are shown. #, P<0.05; *, P<0.01

comparing XIAP inhibitor to solvent.

Figure 6. XIAP inhibitor cooperates with TRAIL receptor antibodies to induce

apoptosis in primary pancreatic carcinoma cells.

A, Surface expression of TRAIL receptors was analyzed by flow cytometry in primary

cultured pancreatic carcinoma cells (ULA).

B, Expression levels of cIAP1, cIAP2, XIAP, survivin and β-actin was assessed by Western

blotting in primary cultured pancreatic carcinoma cells (ULA).

C, Primary cultured pancreatic carcinoma cells (ULA) were treated for 48h with indicated

concentrations of TRAIL-R1 Mapatumumab (Mapa), Lexatumumab (Lexa) or Mapatumumab

and Lexatumumab (Mapa + Lexa) and/or 10 μM XIAP inhibitor 3 or DMSO. Cell viability






32

was determined by MTT assay and is expressed as percentage of untreated controls. Mean +

SEM of three independent experiments performed in triplicate are shown; #, P<0.05; *,

P<0.01 comparing XIAP inhibitors to solvent.

D, PancTu1 cells were seeded on the CAM of chicken embryos, allowed to establish and

treated for 3 d with 0.25 µg Mapatumumab (Mapa) or Lexatumumab (Lexa) in the presence

or absence of 10 µM XIAP inhibitor 3. The CAM was excised on day 4, fixed and H&E

stained. Tumor cell area was determined as described in Materials and Methods and is

presented as percent of total tumor area. Mean + SEM of a representative experiment is

shown; #, P<0.05; *, P<0.01 comparing XIAP inhibitors to solvent.




Fig. 1A




TRAIL-R1 TRAIL-R2 TRAIL-R3 TRAIL-R4

Fig. 1B

PaTu8988s

MiaPaCa2

PaTu8988t

Panc1

BxPC3

cell

coun

t

Panc98

c

T3M4

PaTu8902PaTu8902

fluorescence intensity




Fig. 2PancTu1

80

100

80

100

PaTuII PaTu8902

80

100

A

Colo357

0

20

40

60

0 0.03 0.1 0.3 1 3

BxPC3

0

20

40

60

0 0.3 1 3 6 10

Panc-1

0

20

40

60

0 0.3 1 3 6 10

20

40

60

80

100

20

40

60

80

100

20

40

60

80

100

lity

(%

)

DanG

60

80

100

0

0 0.01 0.03 0.1 0.3 1

0

0 0.03 0.1 0.3 1 3 6 10

0

0 0.03 0.1 0.3 1

T3M4

60

80

100

cell

viab

il

0

20

40

0 0.3 1 3 6 10

100

MiaPaCa2 PaTu8988t

100 100

ASPC1

0

20

40

0 0.1 0.3 1 3 6 10

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

0 0.3 1 3 6 10

0

0 0.3 1 3 6 10

DMSO/Mapa XIAP inh. 2/Mapa

DMSO/Lexa XIAP inh. 2/Lexa

DMSO/Mapa + Lexa XIAP inh. 2/Mapa + Lexa

0

0 0.3 1 3 6 10

PaTu8988s

60

80

100

Panc98

60

80

100

agonistic TRAIL-R mAB (µg/ml)

0

20

40

0 0.3 1 3 6 10

0

20

40

0 0.3 1 3 6 10




80

100

(%)

DMSO

80

100

(%)

BPancTu1 PaTuII

** *

* *

* **

* *

0

20

40

60

0 0.03 0.1 0.3 1 3

Mapa (µg/ml)

cell

viab

ility

( DMSO

XIAP inh. 3

0

20

40

60

0 0.3 1 3 6 10

Mapa (µg/ml)

cell

viab

ility

Mapa (µg/ml) Mapa (µg/ml)

40

60

80

100

viab

ilit

y (%

)

40

60

80

100

viab

ility

(%

)

* *#

* * *

0

20

0 0.03 0.1 0.3 1 3

Lexa (µg/ml)

cell

v

0

20

0 0.3 1 3 6 10

Lexa (µg/ml)

cell

100 100* **

0

20

40

60

80

cell

viab

ility

(%

)

0

20

40

60

80

cell

viab

ility

(%

)** *

** *

*

00 0.03 0.1 0.3 1 3

Mapa + Lexa (µg/ml)

00 0.3 1 3 6 10

Mapa + Lexa (µg/ml)

PancTu1 PaTuII

2030405060708090

100

cell

viab

ility

(%

) 30 µM10 µM3 µM1 µM0.3 µM0 µM

2030405060708090

100110

cell

viab

ility

(%

)

CPancTu1 PaTuII

01020

0 0.03 0.1 0.3 1 3

Mapa (µg/ml)

c

01020

0 0.3 1 3 6 10

Mapa (µg/ml)




60

70 DMSO/MapaDMSO/LexaDMSO/Mapa + Lexa 60

70

A

Fig. 3

PancTu1 PaTuII

**

**

0

10

20

30

40

50

apo

pto

sis

(%)

XIAP inh. 3/Mapap

XIAP inh. 3/LexaXIAP inh. 3/Mapa + Lexa

0

10

20

30

40

50

apo

pto

sis

(%)

A*

*

*# *

*

00 0.03 0.1 0.3 1 3


00 0.3 1 3 6 10


DMSODMSO/Mapa

PancTu1 PaTuII

pto

sis

(%)

30

40

50

60

70

30

40

50

60

70

pto

sis

(%)

XIAP inh. 3

p

XIAP inh. 3/Mapa

DMSO/Lexa

XIAP inh. 3/Lexa

DMSO/Mapa + Lexa

XIAP inh. 3/Mapa + Lexa

B

**

*

*

*

*ap

op

0

10

20

24 48 72

time (h)

0

10

20

30

24 48 72

time (h)

apo

p

C medium DMSOXIAP inh. 3

medium

medium DMSOXIAP inh. 3

medium

PancTu1 PaTuII

Mapa

Lexa

Mapa

Lexa




Fig. 4

A100100

PancTu1 PaTuII

40

60

80

cell

viab

ility

(%)

40

60

80

cell

viab

ility

(%)

****

*

*

** *

0

20

Mapa Lexa TRAIL R1-5 R2-6

0

20

Mapa Lexa TRAIL R1-5 R2-6

XIAP inh. 3 +- +- +- +- +- XIAP inh. 3 +- +- +- +- +-

B

80

100

80

100

PancTu1 PaTuII

**

*

*

20

40

60

80

cell

viab

ility

(%)

20

40

60

80

cell

viab

ility

(%) **

0

Mapa Lexa

+- +- +- +-

0

crossl.

Mapa Lexa

+- +- +- +-++ + +----XIAP inh. 3

crossl.

XIAP inh. 3 ++ + +----




Fig. 5

A

Casp-8

Mapa Lexa

DMSO XIAP inh. 30 2 5 12 2

448

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

DMSO XIAP inh. 3 DMSO XIAP inh. 3time (h)

55/53 kD

Casp-3

Casp-8

Pan

cTu1

55/53 kD

32 kD

β-actin

Casp-9

P

48 kD

41 kD

Casp-8

Casp-8

Mapa Lexa

DMSO XIAP inh. 30 2 5 12 2

448

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

2 5 12 24

48

DMSO XIAP inh. 3 DMSO XIAP inh. 3time (h)

55/53 kD

55/53 kD

Casp-9

Casp-3

PaT

uII 32 kD

48 kD

β-actin

Casp 9

41 kD




B Mapa/ DMSOMapa/ XIAP inh. 3Lexa/ DMSO

6

7

7

8

PancTu1 PaTuII

**

Lexa/ XIAP inh. 3

fold

indu

ctio

n of

ca

spas

e-3

activ

ity

1

2

3

4

5

6

fold

indu

ctio

n of

ca

spas

e-3

activ

ity

1

2

3

4

5

6

7

* *

* * **

*

#

#

time (h)

02 5 12

time (h)

0

1

2 5 12

CPancTu1 PaTuII

C

20

40

60

80

100

20

40

60

80

100

cell

viab

ility

(%)

20

40

60

80

100

20

40

60

80

100

cell

viab

ility

(%)

* #* *

* *

XIAP inh. 3zVAD.fmk

Mapa Lexa

0

20

0

20

+

0

20

-0

20

Mapa Lexa

+++

+ ++

--

--

--+

-+ +--+-- +

+ +--+-- +




A

Fig. 6

tTRAIL-R1 TRAIL-R2 TRAIL-R3�� TRAIL-R4

fluorescence intensity

cell

cou

nt

60

80

100

y (%

)

B C

cIAP1 70 kD

0

20

40

60

cell

viab

ility

DMSO/Mapa

XIAP inh. 3/Mapa

DMSO/Lexa

XIAP inh. 3/Lexa

DMSO/Mapa + Lexa

XIAP inh. 3/Mapa + Lexa

cIAP2

XIAP

survivin

β-actin

72 kD

53 kD

16 kD

41 kD

0 0.03 0.1 0.3 1 3


D70

#

#

30

40

50

60

r ce

ll ar

ea (

%)

0

10

20tum

o

XIAP inh. 3

mapa+ + +

++-- -- -

-mapa

lexa+

++

+- -

- -- -- -




Tab. 1. TRAIL receptor expression in primary pancreatic carcinoma samples

Results of immunohistochemistry were scored as “negative”, n; “weakly positive”, wp; “positive”, p; “strongly positive”, sp, or in cases of staining heterogeneity as p/n. dpc, ductal pancreatic carcinoma; pc, pancreatic carcinoma; c., cell.




Table 2: Synergistic induction of apoptosis by XIAP inhibitor and Mapatumumab.

Cell line

Concentration

(µg/ml)

CI value

XIAP inh. 3

PancTu1 0.03 0.031

0.1 0.008

0.3 0.002

1 0.002

3 0.001

PaTuII 0.3 0.194

1 0.08

3 0.034

6 0.036

10 0.029

Combination index (CI) was calculated as described in Materials and Methods for apoptosis induced by combined treatment of pancreatic cancer cells cells for 72h with indicated concentration of Mapatumumab and 10 µM XIAP inhibitor 3. CI < 0.9 indicates synergism.




Published OnlineFirst October 12, 2010.Clin Cancer Res Dominic Stadel, Caroline Ref, Marion MacFarlane, et al. enhanced by XIAP inhibitorsreceptor 1 in pancreatic carcinoma cells and profoundly TRAIL-induced apoptosis is preferentially mediated via TRAIL

Updated version

10.1158/1078-0432.CCR-10-0985doi:

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