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EGFR-pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells

Niels Eckstein1, Kati Servan1, Luc Girard2, Di Cai2, Georg von Jonquieres1, Ulrich Jaehde3, Matthias U. Kassack4, Adi F. Gazdar2, John D. Minna2, and Hans-Dieter Royer1

1Stiftung caesar, Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany, 2Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA, 3Department of Clinical Pharmacy, University of Bonn,

An der Immenburg 4, 53121 Bonn, Germany, 4Pharmaceutical Biochemistry, Institute of Pharmaceutical and Medicinal Chemistry, University of Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf,

Germany, Running title: amphiregulin as a novel mechanism of cisplatin resistance

Address correspondence to: Hans-Dieter Royer, M.D., Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany; Tel: +49/228/9656 168, e-mail: royer@caesar.de The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin resistant MCF-7 cells and determined the functional status of EGFR, MAPK, and AKT signalling pathways by phospho-receptor tyrosine kinase- and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signalling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL2 expression. We investigated the expression of genes encoding the ligands for the ERBB signalling cascade and found a selective upregulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGF-receptor (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time and knock down of amphiregulin expression by specific siRNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate generality and importance of our findings, we examined amphiregulin expression and cisplatin

resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have, thus, identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.

The use of platinum complexes for the therapy of breast carcinomas is an emerging new treatment modality that has recently been introduced into the clinic, reviewed in (1). Breast cancer is a family of diseases that consists of major categories including HER-2-positive breast cancer, “triple-negative” tumors that are ER-negative, PR-negative and HER-2-negative, and hormonally sensitive breast cancers. The estrogen-receptor expressing (ER-positive) breast cancers are the most prevalent (2). For the therapy of HER2-overexpressing metastatic breast cancer, platinum complexes have been used in combinbation with paclitaxel and trastuzumab, a humanized monoclonal IgG1 that binds the extracellular domain of the ERBB2 (HER-2/neu) receptor (3). For the treatment of HER-2 positive locally advanced breast cancer, a combination of docetaxel, cisplatin and trastuzumab have been used as primary systemic therapy (4). Several ongoing phase II studies explore the use of platinum salts for the therapy of breast cancer including “triple negative” (ER-, PR- and HER-2-negative) breast carcinomas (www.clinicaltrials.gov).

http://www.jbc.org/cgi/doi/10.1074/jbc.M706287200The latest version is at JBC Papers in Press. Published on October 17, 2007 as Manuscript M706287200

Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cisplatin enters the cells predominantly by passive diffusion, where it undergoes aquation to form [Pt(NH3)2Cl(OH2)]+ and [Pt(NH3)2(OH2)2]2+ (5). Cisplatin functions as a bivalent electrophile predominantly inducing formation of 1,2-intrastrand d(GpG) DNA cross-links (6). Although many cellular components interact with cisplatin, DNA is thought to be the primary biological target of the drug (7). Recently, it was demonstrated that the epidermal growth factor receptor (EGFR) becomes phosphorylated at threonine 669 (T669) by p38 MAPK when non-resistant MCF-7 breast cancer cells were exposed to cisplatin (8). Thus, the EGFR signaling pathway is involved in cellular defense against the toxic effects of cisplatinum compounds. The ERBB receptor-ligand network comprises a total of four receptors, EGFR (ERBB1), ERBB2 (HER-2) ERBB3, and ERBB4, and multiple ligands reviewed in (9). Ligands that bind to the ERBB receptor family, include EGF, transforming growth factor alpha (TGFA), heparin-binding EGF-like ligand (HB-EGF), amphiregulin (AREG), betacellulin (BTC), epiregulin (EPR), epigen (EPGN), and neuregulin (NRG) family members (10). It is known that an extraordinary variety of different isoforms are produced from the NRG1 gene by alternative splicing. These isoforms include heregulins (HRGs), glial growth factors (GGFs), and sensory motor neuron-derived factor (SMDF). The NRG1 gene is located on chromosome 8 and additional neuregulin genes were identified on chromosome 5 (NRG2), 10 (NRG3) and 15 (NRG4) (11). It is well established that the ERBB receptor ligands activate distinct subsets of ERBB receptors and differ in their biological activities (12). The EGFR signalling system is connected to a variety of other related pathways and a comprehensive pathway map has been constructed based on published scientific papers (13). The development of cellular resistance to anticancer drugs is a dynamic biological process of high complexity. To better understand this clinically important issue, novel approaches like systems biology are needed. To study cellular mechanisms of resistance to cisplatin, we utilized ER-positive MCF-7 breast cancer cells as a model system. We selected cisplatin resistant MCF-7 breast cancer cells by exposure to sequential

cycles of cisplatin that mimic the way the drug is used in the clinic. We systematically investigated the EGFR signalling system and related pathways, and identified autocrine amphiregulin as a novel molecular mechanism that confers resistance to cisplatin. Examination of a panel of human breast cancer cells revealed that high levels of amphiregulin are associated with resistance to cisplatin thus demonstrating the generality of our findings.

Experimental Procedures Cell Culture and Preparation of Cell Lysates. HCC1419 breast cancer cells were purchased from a vendor affiliated with the ATCC (LGC Promochem GmbH, Wesel, Germany). Cells were grown at 37°C under humidified air, supplemented with 5% CO2 in phenol red-free DMEM (Biochrom AG, Berlin, Germany) containing 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine. Cells were grown to 80% confluency in T-75 cell culture flasks (Corning Life Sciences GmbH, Wiesbaden, Germany). To prepare cell lysates, culture flasks were rinsed two times with ice cold PBS. The following steps were carried out at 4°C. Cells were lysed at a density of 1 x 107 cells/ml in lysis buffer (1 mM EDTA, 0.005% Tween 20, 0.5% Triton X-100, 10 mM NaF, 150 mM NaCl, 20 mM β-glycerophosphate, 1 mM DTT, 25 µg/ml pepstatin, 3 µg/ml aprotinin in PBS, pH 7.2-7.4) for 15 min on ice. Lysates were centrifuged (4000 g, 5 min) and supernatants were transferred into clean test tubes. Total protein content was assessed using a UV-Spectrophotometer (NanoDrop® ND-1000, Kisker, Steinfurt, Germany). Protein lysates (2 µl) were measured at 280nm. The background of the lysis buffer was determined in a separate reaction and subtracted from protein readings. Aliquots of cell lysates were frozen at –80°C. MTT Cell Survival Assay. Cell-survival was quantified by the MTT-assay which is based on the formation of formazane crystals from tetrazolium by living cells. MCF-7 cells were incubated in 24-well plates in 1 ml DMEM. Incubation was terminated by aspirating the media and 300 µl MTT solution (5 mg/ml in PBS) were added to each well. Formazane formation was

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terminated after 15 min by removing the MTT solution. Subsequently 700 µl DMSO were added to each well to solubilize formazane and the formazane containing samples were transferred to a new 24-well plate and measured at 590 nm in a microplate-reader. To measure cell survival after eposure to cisplatin, MTT assays were performed exactly as described above. The cisplatin concentrations and experimental details are described in the text and the legends of the corresponding figures. To measure cell survival after inhibition of amphiregulin by a neutralizing antibody (AF 262, R&D Systems, Wiesbaden, Germany), we added 1µg/ml of the polyclonal antibody to the tissue cultur 1h before the addition of cisplatin and MTT assays were performed as described above. Matrigel Invasion Assay. To determine invasive potential of MCF-7 CisR cells, we utilized the CytoSelectTM 24-well cell migration and invasion assay, colorimetric format (Cell Biolabs Inc. San Diego, CA, USA). The assay was done exactly as described in the invasion assay protocol. In brief, cells were serum-starved for 24h and subsequently they were seeded into the upper chamber onto a rehydrated basement membrane covering a matrigel preparation with a diameter of 8µm. Cells were allowed to invade toward 10% FCS for 24 h. Invaded cells on the bottom of the membrane were stained and quantified as described in the assay protocol. The assay was repeated several times (n=4). Signaling Pathway Analysis. To investigate signaling pathways we used the Proteome ProfilerTM Arrays (R&D Systems, Wiesbaden, Germany). For the analysis of ERBB phosphorylation, a human phospho-RTK antibody array was used. The human phospho-RTK antibody array is a nitrocellulose membrane where 42 different anti-RTK Abs have been spotted including 4 positive controls and 5 negative controls, which are spotted in duplicate. Positive controls are phosphorylated tyrosine kinase receptors, which are recognized by the anti-RTK Abs. For the analysis of serine/threonine kinases, a human phospho-MAPK antibody array was used. The human phospho-MAPK array is a nitrocellulose membrane where 21 different anti-kinase Abs have been spotted including 3 positive controls and 6 negative controls, which

are spotted in duplicate. Positive controls are phosphorylated proteins, which are recognized by the anti-kinase Abs. To conduct a Proteome ProfilerTM Array-experiment the appropriate cells were rinsed twice with PBS and NP-40 lysis buffer was added at density of 1 x 107 cells/ml. Cell lysates were gently rocked for 30 min at 4°C and then centrifuged at 14.000 x g for 5 min (4°C), the supernatants were frozen at -80°C. A total of 250 µg protein was used for each array. To prevent unspecific protein-binding, arrays were blocked using 2% BSA in PBS for 1 h at room temperature. Subsequently cell lysates were diluted with PBS containing 2% BSA and the arrays were incubated with the diluted cell lysates overnight at 4°C. The arrays were then washed 3 times for 10 min with a wash buffer as specified by the manufacturer. Processing of the arrays differs slightly for phospho-RTK and phospho-MAPK antibody arrays. To process phospho-RTK antibody arrays, they were incubated with HRP-conjugated mouse anti-phospho-tyrosine Ab for 2 h at room temperature. To process phospho-MAPK antibody arrays, they were incubated with a biotinylated detection antibody cocktail for 2 h at room temperature, followed by a washing step and incubation with a streptavidin-HRP conjugate (30 min, room temperature). After another washing step both types of arrays were processed using a luminol-based reagent, which is used in combination with HRP-conjugated secondary antibodies (WesternGlo®, R&D Systems, Wiesbaden, Germany). Subsequently, arrays were exposed to X-ray films (5-20 min) and developed under standard conditions. Please note that all array experiments were carried out in triplicate. Immunoblot Analysis. Cell lysates (30 µg protein) were loaded per lane and size fractionated on a 12% SDS-PAGE. Fractionated proteins were transferred to a PVDF membrane using established protocols. To control that equal amounts of protein were loaded in each lane, the membranes were stained with Ponceau S (Sigma-Aldrich Chemie, Steinheim, Germany). For immunoblotting we used a polyclonal affinity-purified goat Ab specific for p53 at a concentration of 1 µg/ml (AF1355, R&D systems, Wiesbaden, Germany) and a polyclonal affinity-purified goat Ab specific for p21 at a concentration of 1 µg/ml (AF1047,

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R&D systems, Wiesbaden, Germany). As secondary antibody we used a horseradish peroxidase conjugated antibody that detects total goat IgG (HAF109, R&D systems, Wiesbaden, Germany). The secondary antibody was used at a 1:2500 fold dilution. Western Blots were processed with the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK) and exposed to X-ray films. X-ray films were developed using standard conditions. Enzyme-linked Immunoassays, AKT Kinase Activity and BrdU Cell Proliferation Assay. To determine the levels of p53 and p21 in cell lysates, we have used a human total p21 and a human total p53 ELISA kit, and for the detection of amphiregulin in cell culture supernatants we have used a human amphiregulin ELISA kit (R&D Systems, Wiesbaden, Germany). BCL-2 levels in cell lysates were measured using a human BCL-2 ELISA kit (Calbiochem, Darmstadt, Germany). ELISAs were performed as specified by the manufacturer. The activity of AKT kinase in breast cancer cells was determined by an AKT kinase activity assay based on a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal Ab that recognizes the phosphorylated form of the substrate (Stressgen Bioreagents, Victoria, Canada). The kit was used in accordance with the manufacturers recommendations. Cell proliferation was quantified using a cell proliferation ELISA which is based on the measurement of BrdU incorporation during DNA synthesis (Roche Applied Science, Penzberg, Germany). The assay was performed as recommended by the manufacturer. Microarray Analysis with the Agilent System. For gene expression analysis, Agilent 44k whole genome microarray slides were used. RNA samples (500 ng each) were amplified and labeled with CY-3-CTP and CY-5-CTP respectively (NEN Perkin Elmer, Boston, MA, USA) to gain labeled cRNA following a protocol published by the manufacturer. Dye-incorporation ratio (≥10 pmol dye per µg cRNA) was measured using the Nanodrop photometer (Kisker, Steinfurt, Germany). For hybridization, 1 µg CY-3-labeled control and 1 µg CY-5-labeled treated samples were mixed and incubated according to the

manufacturers instructions (Agilent Technologies, Böblingen, Germany). Washing steps were performed for 1 min in 2 x SSPE/0,01% N-lauryl-sulphate and 1 min in 0,01xSSPE/0,01% N-lauryl-sulphate. Finally slides were incubated 1 min in Acetonitril. Completely dried slides were scanned using Agilent Microarray Scanner. Data analysis and interpretation was performed using Rosetta Luminator Software (Rosetta Biosoftware, Seattle, USA).

Affymetrix Microarray GeneChip Expression Analysis. Total RNA (5 µg) was extracted with RNeasy Miniprep (Qiagen) protocol. The quality of the total RNA was checked with denaturing formamide gel electrophoresis, which showed two sharp and distinct bands of 18S and 28S. Quality check was also done by the Agilent Bioanalyzer with graphical analysis showing two distinct peaks of 18S and 28S without additional peaks of degradation. The total RNA was then hybridized onto Affymetrix GeneChip HG-U133 A and B sets according to standard protocols (Affymetrix Microarray Suite User Guide 5.0). Quantification of Amphiregulin mRNA by Real-time RT-PCR.. Total cellular RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. The concentration of purified RNA was determined by the Agilent 2100 bioanalyzer using RNA 6000 Nano Chips (Agilent, Waldbronn, Germany). As an internal standard, the ß-Actin gene was chosen. RT-PCR was performed using QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) on DNA Engine Opticon (BioRad, München, Germany). All reactions were performed with 500 ng total RNA in a volume of 25 µl. Thermal cycling conditions were 30 min at 50°C, 15 min at 95°C, followed by 35 cycles of 30 sec 95°C, 30 sec 60°C and 30 sec 72°C finished with a melting curve. Expression levels of amphiregulin and ß-actin were quantified by the ∆∆C(T) method. The primers used were amphiregulin forward (5´-GCTCTTGATACTCGGCTCAG-3´) and amphiregulin reverse (5´-CCCG-AGGACGGTTCACTAC-3´); ß-actin forward (5´-AGAAAATCTGGCACCACACC-3´) and ß-actin reverse (5´-CAGAGGCGTACAGGGATAGC-3´). The amphiregulin mRNA concentration was normalized to β-actin mRNA.

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Amphiregulin Knock Down by siRNA. Transfection of 21 nucleotide siRNA duplexes (Qiagen, Hilden, Germany) for targeting endogenous amphiregulin was carried out using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany). Transfection was performed in suspension and cells were plated at 1 x 104 per well into 24 well plates. The final volume of 0.5 ml contained 33 nM of both siRNA 1 and siRNA 2 (siRNA 1: CCACAAAUACCUGGCUAUAdTdT; siRNA 2: AAAUCCAUGUAAUGCAGAAdTdT) and 1 µl Lipofectamine 2000. As described elsewhere, highest efficiencies in silencing target genes are obtained by using mixtures of siRNA duplexes targeting different regions of the gene of interest (14). As control we used a nonsilencing siRNA (AllStars RNAi control, Quiagen, Hilden, Germany. In general, siRNA treated cells were analyzed 72 h after transfection. Fitting of Concentration/Effect Curves. Indicated are mean values ± SEM. Concentration/effect curves were fitted to data by nonlinear regression analysis using the following four-parameter logistic equation

Hn

x50

10IC1

MinMaxMinY⎟⎠⎞

⎜⎝⎛+

−+=

X refers to the drug concentration; Y refers to the response. The parameters “Max” and “Min” refer to the upper and lower plateau of the sigmoidal curve, respectively. IC50 denotes the X-value at the inflection point of the sigmoidal curve, and nH is the slope factor of the curve. Statistical Methods. Assays were performed in triplicate unless otherwise indicated. The data are reported as mean ± SEM. Statistical significance was assessed by two-tailed Student t-test for single comparison and ANOVA for multiple comparisons, respectively.

RESULTS Generation and Characterization of Cisplatin Resistant MCF-7 Breast Cancer Cells. Hormone receptor-positive breast cancer is the most prevalent form of the disease (15). In this study, we used ER-positive MCF-7 mammary carcinoma cells as a representative model system to investigate cellular mechanisms of resistance to cisplatin. MCF-7 cells were originally derived

from a malignant pleural effusion from a postmenopausal woman with metastatic infiltrating ductal carcinoma of the breast (16). After transplantation into nude mice, MCF-7 cells form tumors with a very low potential of visceral metastasis (17). To generate cisplatin resistant MCF-7 breast cancer cells, we exposed the cells to sequential cycles of cisplatin using a similar dose that is used for the treatment of women with breast cancer. At a confluency of 80% (approximately 1x 107 cells) the tissue culture medium was removed and new medium containing 3 µM cisplatin was added. This concentration of cisplatin represents an IC50 concentration that was determined empirically using MCF-7 cells. After 8 hours of cisplatin exposure the cells were washed 3 times with DMEM and cultivated as described. During the first two months, MCF-7 cells were treated with 8 cycles of cisplatin at weekly intervals. After that, the cells were treated with cisplatin in monthly intervals. After 6 months the extent of cisplatin resistance was quantified by a MTT cell survival assay, which is shown in Fig. 1A. In the MTT cell survival assay non-resistant MCF-7 cells were used as control that had also been cultivated for 6 months. Nonlinear regression curves were fitted to the data points following four-parameter logistic equation as outlined in experimental procedures. The resistance factor was calculated 3.3 from cell survival curves (p < 0.05). Cisplatin resistant MCF-7 cells that represent an endpoint of our cisplatin treatment regimen were denoted MCF-7 CisR in this work. To test whether cellular resistance to cisplatin is associated with cross resistance we analyzed the anthracycline doxorubicin, which is a prominent chemotherapeutic drug for the treatment of breast cancer(18). We treated MCF-7 and the cisplatin-resistant MCF-7 CisR cells with increasing concentrations of doxorubicin and determined cell survival by the MTT assay (Fig. 1B). The figure shows that MCF-7 CisR cells developed a partial cross resistance to doxorubicin. Next, we determined the proliferation rates of MCF-7 CisR cells in comparison to non-resistant MCF-7 cells. To this end, 6.5 x 104 cells were seeded into individual wells of 6-well plates, cultivated for 72 h and cell numbers were determined using a cell counter. The population doubling time of MCF-7 cells was 37 h. In

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contrast, it was 19 h in the resistant cells. This result is highly significant (P < 0.001). Similar results were obtained by a proliferation assay that measures BrdU incorporation into DNA in S-phase of the cell cycle (Table 1). Thus, the cisplatin resistant state in MCF-7 breast cancer cells is characterized by increased proliferation rates. To test whether cisplatin resistance would affect tumor cell behaviour, we examined the metastatic potential of MCF-7 CisR cells by a matrigel invasion assay which monitors whether the cells have an increased ability to invade the matrix of a reconstituted basement membrane (Fig. 1C). In comparison to MCF-7 cells, the cisplatin resistant MCF-7 CisR have a significantly increased invasive ability. The assay was done several times (N=4). These results demonstrate that the development of a cisplatin resistant phenotype is associated with increased tumor cell aggressiveness. Analysis of the EGFR Signalling Pathway in MCF-7 CisR Cells. It was previously reported that the epidermal growth factor receptor (EGFR) is activated in response to cisplatin in normal and in tumour cells as part of a cellular survival response (19;20). This response can be classified as a cellular defense mechanism which is activated within several hours after exposure to cisplatin. In contrast, it is well established that the development of drug resistance is a long-term, time-dependent process. In order to gain insight into the mechanisms of cisplatin resistance we investigated the epidermal growth factor receptor (ERBB) signalling cascade in MCF-7 CisR cells. To investigate the phosphorylation status of the ERBB receptor family, we used a phospho-receptor tyrosine kinase (phospho-RTK) array. In this assay, monoclonal capture antibodies, specific for a variety of RTKs, have been spotted in an array format. Phosphorylation of ERBB subunits is subsequently detected by a pan anti-phospho-tyrosine Ab conjugated to horseradish peroxidase. In non-resistant cells the EGFR was phosphorylated at a low level. In contrast, in resistant MCF-7 CisR cells both the EGFR and ERBB2 receptors were strongly phosphorylated (Fig. 2A). The phospho-RTK array detected very low (if any) ERBB3 and ERBB4 phosphorylation in both MCF-7 and MCF-7 CisR cells. Thus, these

receptor subtypes are not activated in cisplatin resistant breast cancer cells. The ERBB signaling pathway is connected to three major mitogen-activated protein kinase (MAPK) pathways and the PI3K/AKT survival pathway (13). The MAPK pathways consist of the ERK1/2 module, the p38 MAPK module, and the JNK module (21). To gain insight into the activities of these MAPK modules in MCF-7 CisR cells, we investigated the phosphorylation status of these modules by a human phospho-MAPK array. The principle of this assay is that capture antibodies specific for MAPKs have been spotted on nitrocellulose membranes. After incubation with cell lysates, a cocktail of phospho-site specific biotinylated antibodies is used to detect phosphorylated MAPKs. The phospho-MAPK array shows that ERK1 (MAPK3) phosphorylation was notably increased in the resistant MCF-7 CisR cells (Fig. 2B). The phospho-MAPK array detects phosphorylation of ERK1 at the T202/Y204 phosphorylation site. In contrast, ERK2 (MAPK1) phosphorylation was very low in both non-resistant and cisplatin resistant MCF-7 cells. The phospho-MAPK array detects phosphorylation of ERK2 at the T185/Y187 phosphorylation site. Next, we investigated the p38 MAPK module. p38 MAPK consist of four isoforms p38-alpha (MAPK14), p38-beta (MAPK11), p38-gamma (MAPK12), and p38-delta (MAPK13). In mammalian cells, the p38 isoforms are strongly activated by environmental stresses and inflammatory cytokines, but not appreciably by mitogenic stimuli (22). The phosphorylation of the p38 MAPK isoforms is mediated by a complex cascade of protein kinases that is illustrated in detail by PhosphoSite® (www.phosphosite.org). The human phospho-MAPK array detects phosphorylation at T180/Y182 (p38-alpha), T180/Y182 (p38-beta), T183/Y185 (p38-gamma), and T180/Y182 (p38-delta). It is evident that the phosphorylation levels of all four isoforms of p38 MAPKs are very similar in MCF-7 and MCF-7 CisR cells (Fig. 2C). Thus, the p38 MAPK module is not activated in cisplatin resistant cells. Next, we investigated the JNK module using the phospho-MAPK array. The JNK family consists of JNK1 (MAPK8), JNK2 (MAPK9) and JNK3 (MAPK10). The JNKs are strongly activated in response to cytokines, UV irradiation,

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growth factor deprivation, and DNA-damaging agents (23). JNK activation requires dual phosphorylation on tyrosine and threonine residues within a conserved TPY motif (24). Like p38 MAPKs, the JNKs are also activated by a complex cascade of kinases (25). The phospho-MAPK array detects phosphorylation of the phosphorylation site T183/Y185 (JNK1), T183/Y185 (JNK2) and T221/Y223 (JNK3). The phospho-MAPK array shows equal albeit very low levels of JNK1, JNK2 and JNK3 phosphorylation in MCF-7 and MCF-7 CisR cells (Fig. 2D). Thus, the JNK module is not activated in MCF-7 CisR cells. The PI3K/AKT cell survival pathway is linked to the EGFR pathway by the docking protein GAB 1, that recruits PI3K in response to EGF stimulation of the EGFR (26). PI3K converts phosphatidylinositole-4,5 biphosphonate (PI(4,5)P2) to (PI(3,4,5)P3), and in consequence AKT1-kinase translocates to the cell membrane and interacts with (PI(3,4,5)P3) via its plekstrin homology domain, being phosphorylated at T308 in the activation loop by phosphoinositide-dependent kinase (PDK) 1 and most likely by the rictor-mTOR complex at S473 (27). Three isoforms of AKT kinases (AKT1, AKT2 and AKT3) have been identified so far. Activation of AKT2 is associated with phosphorylation of T309 and S474, whereas activation of AKT3 is associated with T305 and S472 phosphorylation. The human phospho-MAPK array detects S473 phosphorylation (AKT1), S474 and S472 phosphorylation on AKT2 and AKT3, respectively. Fig. 2E shows, that the levels of AKT phosphorylation are very low in non-resistant MCF-7 cells confirming data from the literature (28). In contrast, we find pronounced AKT1 phosphorylation on S473 in MCF-7 CisR cells. However, we did not detect increased phosphorylation of AKT2 (S474) and AKT3 (S472) in cisplatin resistant cells. We conclude that selective phosphorylation of AKT1 is a feature of cisplatin resistant MCF-7 breast cancer cells. Inactivation of the p53 Pathway in Cisplatin Resistant MCF-7 Breast Cancer Cells. It has recently been shown that AKT induces nuclear localization of MDM2 and, in consequence, degradation of p53 (29). To quantify the activity

level of AKT1 kinase in MCF-7 CisR cells, we used an AKT kinase activity assay (Fig. 3A). It is evident that the level of AKT kinase activity is strongly increased in cisplatin resistant MCF-7 CisR cells. To analyze p53 protein by immunoblotting we used a mouse mAb specific for human p53 (Fig. 3B). The immunoblot shows that p53 protein is strongly downregulated in MCF-7 CisR cells to a level below detectability (Fig. 3B, lane 2). To quantify p53 in MCF-7 and MCF-7 CisR cells, we utilized a sandwich ELISA that measures human total p53 in cell lysates and found a 90% lower p53 protein level in MCF-7 CisR cells when compared to non-resistant MCF-7 cells (Fig. 3C). Thus, cisplatin resistant cells are characterized by a p53 pseudo-null phenotype as a result of markedly decreased p53 protein expression. Degradation of p53 will ultimately inactivate the p53 pathway (30), which can be monitored by determining p21 expression. We thus investigated p21 expression in MCF-7 and MCF-7 CisR cells by immunoblotting and a sandwich ELISA that measures total p21 in cell lysates (Fig. 3D, Fig. 3E). It can be seen that p21 expression in cisplatin resistant breast cancer cells is drastically reduced. These data indicate that the p53 pathway is not active in resistant MCF-7 CisR cells. It is known that wild-type p53 can bind to BCL2 and neutralize the death-protective function of BCL2 (31). Moreover, p53 is a negative regulator of Bcl-2 expression (32), suggesting that a lack of p53 in MCF-7 CisR cells might be associated with altered levels of BCL2 protein. To determine the levels of BCL-2 in resistant MCF-7 CisR and non-resistant MCF-7 cells, we used a sandwich ELISA that measures human total BCL-2 in cell lysates. While MCF-7 cells express a low level of BCL-2 protein, the cisplatin resistant MCF-7 CisR cells showed highly elevated BCL-2 levels (Fig. 3F). We conclude that both the functional inactivation of p53 and the high levels of BCL-2 in MCF-7 CisR cells are an important facet of acquired cisplatin resistance in these cells. Upregulation of Amphiregulin Gene Expression During the Development of Cisplatin Resistance in MCF-7 Breast Cancer Cells. Next, we wished to investigate activities of genes encoding the known EGFR/ERBB ligands during

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development of cisplatin resistance. For this analysis, a new batch of non-resistant MCF-7 cells was treated by cycles of cisplatin in weekly intervals for a total of six months and mRNA was isolated one week after each treatment cycle. For the isolation of temporally matched control RNAs untreated MCF-7 cells were cultivated in parallel for a total of six months. For gene expression analysis, Agilent 44k whole genome microarray slides were used. Gene expression data were analyzed using the Rosetta Luminator software. We analyzed amphiregulin (AREG), betacellulin (BTC), EGF, epiregulin (EREG), epigen (EPG), HB-EGF, NRG1, NRG2, NRG3, NRG4 and TGFA gene expression. We detected a transient induction of amphiregulin gene expression in response to cisplatin exposure in the 1-week and 3-week time points, but nearly control levels in the 6-week and 8-week time points. We found that the levels of amphiregulin gene expression began to rise again after three months and steadily increased in MCF-7 CisR cells until the endpoint (6 months) of our cisplatin treatment regime (Fig.S1). In contrast to AREG, the transcription of EPGN, BTC, EREG, EGF, HBEGF, TGFA, NRG1 (variant GGF2), NRG1 (variant SMDF), NRG1 (variant HRG- beta 1), NRG1 (variant HRG-gamma), NRG2 (variant 5), NRG2 (variant 3), NRG3 and NRG4 did not change significantly after exposure to cisplatin at any time (data not shown). In fact, only amphiregulin was detectably expressed in MCF-7 cells and the expression levels for all other ERBB ligands were below background. The amphiregulin microarray expression data were verified by RT-PCR and this analysis yielded identical results (Fig. 4A). We conclude that ER-positive MCF-7 breast cancer cells express the amphiregulin gene at a low level with strongly increased expression in MCF-7 CisR cells at later stages of cisplatin resistance development. Sustained Secretion of the Epidermal Growth Factor Receptor Ligand Amphiregulin by MCF-7 CisR Cells in Response to Cisplatin Exposure Then, we analysed whether the upregulation of amphiregulin gene expression in MCF-7 CisR cells translates into increased amphiregulin protein levels. The transmembrane amphiregulin precursor protein consists of 252 amino acids and the biologically active 84 amino acid-long

amphiregulin protein is released from the membrane by proteolytic activity of the metalloproteinase ADAM 17 (also known as tumor necrosis factor alpha-converting enzyme, TACE) (33). To detect secreted (shedded) amphiregulin, we used an ELISA. MCF-7 and MCF-7 CisR cells were exposed to 3 µM cisplatin for 8 h and, after removal of the drug, the tissue culture supernatants were analyzed with the amphiregulin-specific ELISA in 24 h intervals. Amphiregulin secretion was first detected 24 h after cisplatin exposure. This result shows that amphiregulin secretion occurs as a response to cisplatin treatment. Moreover, the amphiregulin-specific ELISA detected a strong increase in the concentration of secreted amphiregulin over an extended period of time in supernatants of cisplatin treated MCF-7 CisR cells (Fig. 4B, open circles). In this experiment, highest levels of secreted amphiregulin were found 72 h after exposure to cisplatin. In contrast, non-resistant MCF-7 cells did not secrete amphiregulin after exposure to cisplatin. The levels of amphiregulin in supernatants of cisplatin treated non-resistant MCF-7 cells were very low and did not significantly change over a period of 72 h (Fig. 4B, filled circles). Thus, sustained amphiregulin secretion in response to cisplatin treatment is a unique feature of cisplatin-resistant MCF-7 breast cancer cells. Impact of Amphiregulin and AKT-kinase on Cisplatin Resistance. Our data suggested that amphiregulin is directly linked to cisplatin resistance. We thus wished to determine the impact of amphiregulin for the cisplatin resistant state of MCF-7 CisR cells by an amphiregulin knock down experiment. MCF-7 CisR cells were treated with Lipofectamin 2000® and siRNA specifically targeted against the amphiregulin mRNA transcript. As control we used a commercially available nonsilencing siRNA. Knockdown efficiency was controlled by assaying the supernatants of the transfected cells for secretion of amphiregulin 72 h after transfection using a specific ELISA (data not shown). To measure the consequences of amphiregulin inhibition for cisplatin resistance, siRNA transfected MCF-7 CisR cells were analyzed by the MTT cell survival assay. As shown in Fig. 4C, amphiregulin knock down was associated with an

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almost complete reversion of the cisplatin resistant phenotype. The shift factor after amphiregulin inhibition was calculated as 3.8. To examine the role of secreted amphiregulin for cisplatin resistance more directly, we used a neutralizing antibody specific for amphiregulin in MTT cell survival assays. The neutralzing antibody was added to the tissue culture supernatant one hour before the addition of cisplatin. In these experiments (N=2), we found a substantial reversion of cisplatin resistance in MCF-7 CisR cells (Fig. 4D). The shift factor was calculated as 2,35. As amphiregulin activates the ERBB signaling cascade and this pathway is linked to the PI3K/AKT pathway through GAB1, we wished to investiagte the impact of PI3K/AKT signaling on cisplatin resistance of MCF-7 CisR cells. To inhibit the PI3K/AKT kinase pathway we used wortmannin, which irreversably targets PI3K and inhibits its activity (34). MCF-7 CisR cells were cultivated in the presence of 25 nM wortmannin and exposed to increasing amounts of cisplatin. Subsequently, cell viability was determined by the MTT cell survival assay. As a control, MCF-7 CisR cells cultivated without the addition of wortmannin were exposed to increasing amounts of cisplatin and then analyzed by the MTT cell survival assay (Fig. 4E). Inhibition of PI3K caused reversal of cisplatin resistance. This is illustrated by comparing Fig. 4E with Fig. 1, where MTT cell survival assays of non-resistant and MCF-7 CisR cells after exposure to cisplatin are shown. We conclude that activation of the PI3K/AKT signalling pathway is an important event downstream of amphiregulin for the development of cisplatin resistance in MCF-7 breast cancer cells.

Significant Correlation of Amphiregulin Expression with Cisplatin Resistance in Diverse Human Breast Carcinoma Cell Lines. MCF-7 breast cancer cells served as a model system to investigate molecular mechanisms of cisplatin resistance. To test whether our results are of more general importance, we analyzed amphiregulin expression in a panel of 12 human breast carcinoma cell lines. In a second step the sensitivities of these cell lines to cisplatin exposure was measured by MTS cell survival assays. The summary of these data is shown

(Fig.5A). In a final step, we correlated the amphiregulin expression levels with the IC50 values from MTS cell survival assays (Fig.5B). This analysis revealed a correlation coefficient of 0.674, which is highly significant (**p-value: 0.002). Thus, elevated levels of amphiregulin expression indicate a cisplatin resistant phenotype in diverse human breast cancer cells. To verify this experimentally we selected HCC1419 breast cancer cells as a representative example for amphiregulin knock down experiments. The HCC1419 cell line expresses high levels of amphiregulin and was originally established from a nodal positive ductal carcinoma which was treated by prior chemotherapy (35). The MTT cell survival assay shows that the inhibition of amphiregulin by a specific siRNA was associated with a significant reversal cisplatin resistance in HCC1419 breast cancer cells (Fig. 5C). We conclude that amphiregulin is a key factor for cisplatin resistance in human breast cancer cells. To test whether increased amphiregulin expression has an impact on cisplatin resistance in another tumor entity, we correlated the levels of amphiregulin expression with cisplatin resistance in a cohort of lung cancer derived cell lines (n = 43). We found high levels of amphiregulin expression in a large fraction of lung cancer cells (N=43) some of which were highly resistant to cisplatin (Fig. S2 A). However, a statistical analysis of these data did not unveil a significant correlation amphiregulin expression with resistance to cisplatin. The correlation coefficient was calculated as -0,02027 with a nonsignificant p-value (p = 0.895). Future work is needed to clarify whether amphiregulin is linked to anticancer drug resistance in other malignant diseases besides breast cancer.

DISCUSSION ER-positive breast cancers are the most prevalent form of the disease (36). Eventually, in most women, metastatic breast cancer becomes refractory to hormonal treatment and chemotherapy (37). These clinical findings demonstrate that the development of resistance to therapy is a time consuming biological process. Here we have generated cisplatin resistant ER-positive breast cancer cells (MCF-7 CisR) by

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sequential cycles of cisplatin exposure over a period of 6 months. During the first two months the cells received weekly cycles of cisplatin followed by monthly cycles of cisplatin exposure. It is a goal of our work to use systems biology approaches to unveil basic principles of cisplatin resistance. The MCF-7 CisR cells represent the endpoint of our cisplatin treatment regimen and we used these cells to investigate systematically the activities of ERBB and MAPK signalling pathways using phospho-RTK and phospho-MAPK arrays. In MCF-7 CisR cells the EGFR is activated (phosphorylated). It has been reported that the adaptor protein GAB1 (Grb2-associated binder 1) recruits PI3 kinase to the activated EGFR which lacks canonical PI3 kinase binding sites (26). A systems biology approach demonstrated that the essential function of GAB1 is to enhance PI3K/AKT activation and to extend the duration of RAS/MAPK signaling (38). In keeping with this, we have detected selective phosphorylation of ERK1 at the T202/Y204 phosphorylation site and selective phosphorylation of AKT1 at S473. It is important to figure out how these phosphorylation events could be linked to the cisplatin resistant state of MCF-7 CisR cells. Several reports from the literature show that ERK1 and ERK2 have different functions (39). While ERK1 is linked to cell proliferation and the survival of tumor cells (40), ERK2 has been linked to the regulation of cell motility (41). Thus, the activation of ERK1 in MCF-7 CisR cells can contribute to increased cell proliferation and cell survival. It has also been shown that the three AKT isoforms have different functions (42). For example, only AKT1 is required for proliferation, while AKT2 promotes cell cycle exit through p21 binding (Heron-Milhavet). Likewise, knock out mice have shown that loss of Akt1 leads to growth defects whereas loss of Akt2 primarily affects glucose homeostasis (43). Most notably, however, it was found that AKT1 amplification in lung cancer tissues was associated with resistance to cisplatin (44). In line with this, inhibition of PI3K/AKT by wortmannin in MCF-7 CisR cells was associated with an almost complete reversal of the cisplatin resistant phenotype. MCF-7 CisR cells are characterized by elevated levels of AKT kinase activity. It is a goal

of our work to unveil mechanisms of cisplatin resistance by a systematic analysis of selected pathways and we thus focused on signaling downstream of AKT. It is well established that AKT phosphorylates a number of substrates at specific serine and threonine residues including transcription factors, protein kinases, apoptosis regulators and components of the proteasome (45). For example, AKT dependent phosphorylation of MDM2 at S166 and S186 promotes translocation to the nucleus where it negatively regulates p53 function (46;47). MCF-7 cells are wild-type for p53 and MCF-7 CisR cells are characterized by strongly reduced p53 protein levels which are associated with an inactivation of the p53 pathway. It has to be debated whether the reduction of p53 is due to increased cell proliferation or whether the loss of p53 is causing this increase in proliferation. It has been shown that silencing of p53 by siRNA promotes accelerated DNA synthesis (48). MCF-7 CisR cells show accelerated DNA synthesis as determined by BrdU incorporation. In addition, chemotherapeutic drugs including anthracyclins induce p53 expression and p53 dependent apoptosis(49). If the lower levels of p53 and p21 in MCF-7 CisR cells are due to accelerated proliferation one might expect that they are sensitive to other chemotherapeutic drugs. We chose doxorubicin to address this issue and found that MCF-7 CisR cells are partially cross resistant to this drug. This result supports the notion loss of p53 and p21 in MCF-7 cells is responsible for accelerated proliferation and drug resistance. It is evident that inactivation of p53 is an important step for the development of cisplatin resistance as p53 antagonizes BCL-2 function at several levels (50) p53 is also controlling the expression of Bcl-2 at the level of transcription as antisense inhibition of p53 is associated with overexpression of Bcl-2 (51), and transient transfection analysis revealed that wild-type p53 repressed the Bcl-2 full length promoter (52). In MCF-7 CisR cells the downregulation of wild-type p53 protein expression is associated with increased levels of BCL-2 suggesting that both events are interconnected in the resistant cells. In addition, AKT kinase up-regulates Bcl-2 expression through phosphorylation of a cAMP-response element-binding protein. It is well established that BCL-2

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prevents apoptosis induced by most chemotherapeutic drugs (53). The JNK and p38 MAPK modules of the MAPK signalling pathway were not activated in MCF-7 CisR cells. It is established that these two stress-activated modules are directly linked to apoptosis and it is known that apoptosis-signal regulating kinase 1 (ASK-1) acts upstream of these MAPK modules (54). ASK-1 is a substrate of AKT kinase and phosphorylation inhibits ASK-1 function (55). It is conceivable that ASK-1 phosphorylation by AKT1 in MCF-7 CisR cells prevents activation of the JNK and p38 MAPK modules. It is, however, part of future work to elaborate this issue. So far we have discussed the current status of ERBB and MAPK signalling pathways in MCF-7 CisR cells. In the long run, however, we are interested to analyze the process of cisplatin resistance development in a time-resolved fashion. To address this issue, we used Agilent 44k whole genome microarrays and analyzed gene expression profiles during the process of cisplatin resistance development. For microarray analysis, the MCF-7 cells were exposed to cisplatin in weekly intervals over a total period of 6 months. The ERBB pathway is activated by a family of diverse ligands which bind to the ERBB receptor subunits (56;57). These ligands can be defined as the input level of the ERBB signalling pathway. Gene expression profiling revealed that amphiregulin is the only EGFR ligand that was expressed in non-resistant MCF-7 cells. When we analyzed expression of the ERBB ligands in a time-resolved fashion we found that amphiregulin gene expression was transiently upregulated during the first three weeks of cisplatin treatment and returned a level similar as in non-resistant MCF-7 cells in the fourth week. Thereafter the levels of amphiregulin expression were unchanged for the next 8 weeks. However, after 12 weeks of weekly cisplatin treatment amphiregulin expression increased again reaching highest levels after 6 months. Amphiregulin is an exclusive ligand of the EGFR which induces tyrosine phosphorylation and receptor activation (58). Amphiregulin was originally purified from the conditioned media of MCF-7 breast cancer epithelial cells treated with the tumor promoter PMA (59). A comparison between the biological

effects of EGF and amphiregulin reveals distinct differences (60). Amphiregulin increases invasion capabilities of MCF-7 breast cancer cells, and transcriptional profiling experiments revealed that amphiregulin and EGF promote dramatically distinct patterns of gene expression (61;62). Several genes involved in cell motility and invasion were upregulated when nontumorigenic breast epithelial cells were cultivated in the presence of amphiregulin (63). The cytoplasmic tail of the EGFR plays a critical role in amphiregulin mitogenic signalling but is dispensable for EGF signalling (64). Breast cancer cells which were derived from an aggressive inflammatory breast carcinoma overexpress amphiregulin which renders them EGF-independent (65). Escape of dependence on extrinsic proliferative signals is a key event in the evolution of malignant tumors. Clinical investigations revealed that the levels of amphiregulin protein are generally higher in invasive breast carcinomas than in ductal carcinoma in situ (DCIS) or in normal mammary epithelium (66-68). We have used matrigel invasion assays to characterize tumor cell behaviour of MCF-7 CisR cells and found a significantly increased ability to invade and penetrate the basement menbrane which is the essential component of the matrigel invasion assay. These results are in line with published data and they show that drug resistance and tumor aggressiveness are interconnected biological processes. Here we have identified a novel function of amphiregulin for the cisplatin resistant state of MCF-7 breast cancer cells. The initial transient upregulation of amphiregulin expression suggests that this is part of a cellular defence mechanism. The continuous upregulation of amphiregulin expression during the last three months of cisplatin treatment cycles shows that this is part of a cellular resistance mechanism. Our results suggested that amphiregulin could play an essential role for the development of cisplatin resistance. This consideration was tested by amphiregulin knock down experiments. It was possible to reverse the cisplatin resistant state of MCF-7 CisR cells to a large part by siRNA mediated inhibition of amphiregulin expression. Amphiregulin protein is anchored to the cell

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membrane as a 50-kDa proamphiregulin form and is preferentially cleaved by ADAM 17 at distal site within the ectodomain to release a major 43-kDa amphiregulin form into the medium (69). We conclude that MCF-7 cells show persistant alterations of signaling activity in the ERBB and MAPK pathways which are associated with an inactivation of the p53 pathway and BCL-2 overexpression. In addition, during later stages of resistance development the amphiregulin gene is continuously upregulated. This dynamic process enables the resistant cells to respond to cisplatin by sustained secretion of amphiregulin which ultimately provokes full-fledged cisplatin resistance. Here we have used MCF-7 cells as a model to study mechanism of cisplatin resistance. Once a molecular mechanism is unveiled it is mandatory to explore whether this finding is of general importance for the disease. To address this issue we correlated amphiregulin expression levels with

the cisplatin resistant state of a collection of human breast cancer cells. We found a highly significant correlation which demonstrates that human breast cancer cells use amphiregulin as a survival signal to resist exposure to cisplatin. We also analyzed a collection of lung cancer cells which tend to express elevated levels of amphiregulin. However, we did not find a significant correlation between cisplatin resistance and amphiregulin expression. From these data we conclude that it is necessary to systematically investigate different tumor types in order to determine the role of amphiregulin for cisplatin resistance in other malignant tumors besides breast cancer. Future clinical studies will determine the full impact of amphiregulin expression for therapy response and outcome in women with breast cancer.

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Acknowledgement We thank Sybille Wolf-Kuemmeth, Inge Napierski, Norbert Brenner and Barbara Goetz (caesar) for skillful technical assistance and Susanne Koegel, Irina Buss and Dirk Garmann (Department of Clinical Pharmacy, University of Bonn) for helpful discussions. We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (GRK 677/3) and the University of Bonn. Dr. John Minna was supported by an grant from the National Cancer Institute (NCI SPORE P50CA70907) and the Pulitzer Foundation. The authors declare no financial conflict of interests. FIGURE LEGENDS Table 1. Increased proliferation rates of cisplatin resistant MCF-7 breast cancer cells. Proliferation rates were determined by a BrdU incorporation assay. For the determination of population doubling times 6.5 x 104 MCF-7 and MCF-7 CisR cells were seeded into individual wells of a 6-well plate and cultivated for 72h after which the cell numbers were counted (N=5, ***P < 0.001) Fig. 1. Characterization of cisplatin resistant MCF-7 breast cancer cells. (A) The degree of cisplatin resistance was measured by the MTT cell survival assay. 1 x 104 cells were seeded into individual wells of 24-well plates and exposed to increasing concentrations of cisplatin for 72h. The cisplatin containing tissue culture medium was exchanged with MTT solution and incubated for 15 min. Formazane dye was solubilized by DMSO and formazane formation was quantified by absorbance at 590 nm. MCF-7 cells (filled circles), MCF-7 CisR cells (open circles). Curves were fitted to data points as specified in experimental procedures and the factor of resistance was calculated as 3.3. Arrows indicate IC50 concentrations of cisplatin for MCF-7 cells (left arrow) and IC50 concentrations of cisplatin for MCF-7 CisR cells (right arrow) (N=3, *P < 0.05). (B) Partial cross resistance of MCF-7 CisR cells to doxorubicin. The degree of cross resistance was measured by the MTT cell survival assay. 1 x 104 cells were seeded into individual wells of 24-well plates and exposed to increasing concentrations of doxorubicin for 72h. The assay was done as described in (A). MCF-7 cells (filled circles), MCF-7 CisR cells (open circles). Curves were fitted to data points as specified in experimental procedures and the factor of cross resistance was calculated as 2.0. Arrows indicate IC50 concentrations of doxorubicin for MCF-7 cells (left arrow) and for MCF-7 CisR cells (right arrow) (N=3). (C) Determination of invasive ability of MCF-7 CisR cells. The matrigel invasion assay was done following a protocol of the producer. Invaded cells were detected by a fluorometric procedure 24h after seeding into rehydrated matrigel invasion chambers. (N = 4, Students t-test, *p<0.05). Fig. 2. Systematic analysis of ERBB, MAPK and AKT signaling pathway activity in cisplatin resistant MCF-7 breast cancer cells. (A) Analysis of ERBB phosphorylation. Human phospho-RTK antibody arrays were used to measure phosphorylation of the ERBB subunits EGFR (panel P-EGFR), ERBB2 (panel P-ERBB2), ERBB3 (panel P-ERBB3) and ERBB4 (panel P-ERBB4). The experiments were repeated three times with identical results (N=3). Non-resistant MCF-7 cells (MCF-7) are indicated on the left (A-E) and cisplatin-resistant MCF-7 cells (MCF-7 CisR) are indicated on the right (A-E). Phosphorylated RTKs served as positive control (panel P-tyrosine-positive control). (B) Analysis of ERK1 and ERK2 phosphorylation. A human phospho-MAPK array was used to measure ERK1 phosphorylation at T202/Y204 (panel P-ERK1) and ERK2 phosphorylation at T185/Y187 (panel P-ERK2). Phosphorylated peptides derived from threonine/serine kinases, which are recognized by the detection antibodies served as positive controls (panel positive control, B-E). (C) Analysis of p38 MAPK phosphorylation. Phospho-MAPK arrays were used to measure phosphorylation of p38-alpha at T180/Y182 (panel P-p38 alpha), p38-beta at T180/Y182 (panel P-p38 beta), p38-gamme at T183/185 (panel P-p38 gamma), and p38-delta at T180/Y182. (D) Analysis of JNK phosphorylation. Phospho-MAPK arrays were used to measure phosphorylation of JNK1 at T183/Y185 (panel P-JNK1), JNK2 at

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T183/Y185 (panel P-JNK2), JNK3 at T221/Y223 (panel P-JNK3). (E) Analysis of AKT phosphorylation. Phospho-MAPK arrays were used to measure phosphorylation of AKT1 at S473 (panel P-AKT1), AKT2 at S474 (panel P-AKT2) and AKT3 at S472 (panel P-AKT3). Fig. 3. Analysis of AKT-kinase, downstream signaling reveals inactivation of the p53 pathway in MCF-7 CisR cells. (A) Quantification of AKT kinase activity. To measure AKT kinase activity, a solid phase ELISA, which utilizes a specific synthetic peptide as a substrate and a polyclonal antibody that recognizes the phosphorylated form of the substrate was used. (N=3, ***P < 0.001). (B) Detection of p53 protein by immunoblotting (IB) using a polyclonal affinity purified goat Ab specific for p53. MCF-7 cells (lane 1) and MCF-7 CisR cells (lane 2). Lane M, molecular weight marker. p53 is indicated by an arrow (C) Quantification of p53 by a sandwich ELISA that measures human total p53 in cell lysates (N=3, ***P < 0.001) (D) p21 expression indicates p53 pathway activity. Detection of p21 in whole cell lysates of MCF-7 (lane 1) and MCF-7 CisR cells (lane 2) by immunoblotting using a polyclonal affinity purified goat Ab specific for p21. Lane M, molecular weight marker. p21 indicated by an arrow. (E) To quantify the levels of p21 protein a sandwich ELISA that measures p21 in cell lysates was used. (N=3, ***P < 0.001). (F) To quantify BCL-2 expression an ELISA that detects human BCL-2 in cell lysates was used (N=3, ***P < 0.001). Fig. 4. Identification of amphiregulin as key factor for cisplatin resistance in MCF-7 CisR cells. (A) Amphiregulin mRNA expression in the course of cisplatin resistance development. Amphiregulin mRNA levels were measured by real-time quantitative RT-PCR using gene-specific primers for amphiregulin and ß-actin. The amphiregulin PCR products (Ct-value) were normalized to the level of ß-actin (Ct-value). (N=2, *P < 0.05, ***P < 0.001) (B) MCF-7 CisR cells respond to cisplatin exposure with sustained secretion of amphiregulin. MCF-7 CisR cells (open circles) were exposed to 3 µM cisplatin for 8 h. After replacement of the cisplatin containing medium with DMEM an amphiregulin ELISA was performed on cell culture supernatants (100 µl) at the indicated time points. (N=3, *P < 0.05, ***P < 0.001; n = 3). Non-resistant MCF-7 cells (filled circles) were exposed to an IC50-dose of cisplatin (3 µM) for 8h. After replacement of the cisplatin containing medium with DMEM an amphiregulin ELISA was performed on cell culture supernatants (100 µl) at the indicated time points (N=3). (C) Regulation of cisplatin resistance by amphiregulin. To inhibit amphiregulin expression, MCF-7 CisR cells were transfected using Lipofectamin 2000® and an amphiregulin-specific siRNA. To determine the impact of amphiregulin inhibition on cisplatin resistance 1 x 104 siRNA transfected MCF-7 CisR cells were seeded into indidvidual wells of a 24-well plate and exposed to increasing concentrations of cisplatin. After 72 h MTT cell survival assays were used to measure the degree of cisplatin resistance. siRNA transfected MCF-7 CisR cells (filled stars), nonsilencing siRNA transfected MCF-7 CisR cells (filled triangles), untreated MCF-7 CisR cells (open circles). Curves were fitted to data points as specified in experimental procedures and the shift factor caused by amphiregulin silencing was calculated as 3.8. Arrows indicate the IC50 concentration of cisplatin for MCF-7 CisR cells transfected with the nonsilencing siRNA (right arrow) and the IC50 concentration of cisplatin for siRNA transfected MCF-7 CisR cells (left arrow). (N=2, *P < 0.05). (D) Functional inhibition of secreted amphiregulin by a neutralizing antibody. MTT cell survival assay of MCF-7 CisR cells after exposure to cisplatin in the presence of a neutralizing antibody specific for amphiregulin. The antibody (1µg/ml) was added to the tissue culture 1h before the addition of cisplatin. MCF-7 CisR cells (open circles), MCF-7 CisR cells in the presence of the neutralizing antibody (filled stars). (N=2, *P < 0.05).(E) Impact of the PI3K/AKT kinase pathway for the cisplatin resistant phenotype of human breast cancer cells. MCF-7 CisR cells were cultivated in the presence of 25nM wortmannin to inhibit the PI3K pathway. Wortmannin was added 30 min prior to addition of cisplatin (filled triangles), MCF-7 CisR cells without the addition of wortmannin (open circles). The cells were exposed to increasing concentrations of cisplatin (1-100 µM) and the degree of cisplatin resistance was determined after 72 h by the MTT cell survival assay. Curves were fitted to data points as specified in experimental procedures and the wortmannin-dependent shift factor was calculated as 4.4. Arrows

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indicate the IC50 concentration of cisplatin for MCF-7 CisR cells (right arrow) and the IC50 concentration of cisplatin for MCF-7 CisR cells the in the presence of 25 nM Wortmannin (left arrow). N=3, *P < 0.05. Fig.5. Amphiregulin expression in diverse human breast cancer cells correlates significantly with cisplatin resistance. (A) Summary of human breast cancer cells, cisplatin IC50 values and amphiregulin expression levels. IC50 values were detrmined by MTS cell survival assays (N=3-6) and amphiregulin expression was determined by Affymetrix microarray analysis. (B) Correlation of cisplatin IC50 values from MTS viability assay with amphiregulin gene expression. The correlation coefficient was calculated 0.674 (**P = 0.002). (C) Regulation of cisplatin resistance by amphiregulin in HCC1419 cells. Transfection procedure and MTT assay were carried out exactly as described above (Fig. 4C). siRNA transfected HCC1419 cells (stars), nonsilencing siRNA transfected HCC1419 cells (filled triangles), untreated HCC1419 cells (open circles). Curves were fitted to data points as specified in experimental procedures and the shift factor caused by amphiregulin silencing was calculated as 3.4. Arrows indicate the IC50 concentration of cisplatin for HCC1419 cells transfected with the nonsilencing siRNA (right arrow) and for siRNA transfected HCC1419 cells (left arrow). (N=2, *P < 0.05).

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Table 1

Relative BrdU

incorporation

[%]

Number of cells

[x 104]

Population

doubling

time

Mean SEM n Mean SEM n

MCF-7 100 12.7 3 27.5 2.8 6 37h

MCF-7 CisR 192 26 3 119.6 10.2 5 19h

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Fig. 1

0

50

100

0.01 0.1 1 3 10 100 1000-∞

MCF-7MCF-7 CisR

A

Cisplatin [µM]

Cel

l sur

viva

l [%

]

0

50

100

MCF-7MCF-7 CisR

0.01 0.1 0.3 1 10-∞

B

Doxorubicin [µM]

Cel

l sur

viva

l [%

]

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MCF-7 MCF-7 CisR0

50

100

150

200 *C

Inva

ded

cells

[%]

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Fig. 2

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Fig. 3

MCF-7 MCF-7 CisR

100

150

200

***

A

Rel

ativ

e AK

T ki

nase

act

ivity

[%] o

f unt

reat

ed M

CF-

7

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MCF-7 MCF-7 CisR0

100

200

300***

F

BCL-

2 pr

otei

n [%

] of M

CF-

7

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Fig. 4

0 1 2 3 4 6 7 8 12 16 20 24

0

10

20

30

****

******

A

No of treatment cycles [weeks]

Amph

iregu

lin g

ene

expr

essi

onfo

ld o

ver b

asal

rela

tive

toβ-

actin

0 24 48 72

0

1

2

3

4

MCF-7MCF-7 CisR

B

*

******

Time [h]

Am

phire

gulin

sec

retio

n [n

M]

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0

50

100MCF-7 CisR, untreated controlnon silencing siRNAAmphiregulin siRNA

0.1 1 3 10 100 1000-∞

MCF-7 CisR transfection

C

Cisplatin [µM]

Cel

l sur

viva

l [%

]

0

50

100

MCF-7 CisR + neutr. AbMCF-7 CisR

0.003 0.03 0.3 1 3 10 30-∞

D

Cisplatin [µM]

Cel

l sur

viva

l [%

]

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0

50

100

-∞ 0.3 1 3 10 100

Wortmannin 25nMMCF-7 CisR

E

Cisplatin [µM]

Cel

l sur

viva

l [%

]

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Fig. 5

A

Breast cancer

Cell Line

IC50 from MTS assay

Cisplatin [µM]

Amphiregulin gene expression log 2

from Affymetrix array

HCC1143 4,8 7,986

HCC1419 12,2 11,872

HCC1569 3,4 6,150

HCC1954 8,5 9,289

HCC2218 14,7 8,928

HCC2688 1,4 5,792

HCC3153 1,4 6,458

HTB122 1,2 5,333

HTB126 6,9 5,873

HTB131 2,0 9,005

HTB24 2,6 6,487

MDA-MB 231 4,1 6,932

5 6 7 8 9 10 11 12 13

0

5

10

15

20B

Amphiregulin gene expression

IC50

Cis

plat

in [µ

M]

MTS

sur

viva

l ass

ay

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0

50

100HCC1419, untreated controlnon silencing siRNAAmphiregulin siRNA

0.01 0.1 1 3 10 100-∞

HCC1419 transfectionC

Cisplatin [µM]

Cel

l sur

viva

l [%

]

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Matthias U. Kassack, Adi F. Gazdar, John D. Minna and Hans-Dieter RoyerNiels Eckstein, Kati Servan, Luc Girard, Di Cai, Georg von Jonquieres, Ulrich Jaehde,

resistance of human breast cancer cellsEGFR-pathway analysis identifies amphiregulin as a key factor for cisplatin

published online October 17, 2007J. Biol. Chem. 

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