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P53 and PTEN Expression Contribute to the Inhibition of EGFR Downstream Signaling Pathway by Cetuximab

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P53 and PTEN Expression Contribute to the Inhibition of EGFR Downstream Signaling Pathway by Cetuximab Cancer Gene Therapy (2009) 16, 498–507 r 2009 Nature Publishing Group All rights reserved 0929-1903/09 $32.00 www.nature.com/cgt ORIGINAL ARTICLE P53 and PTEN expression contribute to the inhibition of EGFR downstream signaling pathway by cetuximab S Bouali1, A-S Chre´tien1, C Ramacci1, M Rouyer1, S Marchal2, T Galenne3, P Juin3, P Becuwe4 and J-L Merlin1 1Centre Alexis Vautrin, Unite´ de Biologie des Tumeurs, EA ‘‘Predicther’’ Nancy Universite´, Vandœuvre-le`s- Nancy cedex, France; 2Centre Alexis Vautrin, CRAN UMR 7039 CNRS-UHP-INPL, Vandœuvre-le`s-Nancy cedex, France; 3INSERM U601, CHU de Nantes, Institut de Biologie, Nantes, France and 4Laboratoire de Biologie Cellulaire, Faculte´ des Sciences, EA ‘‘Predicther’’ Nancy Universite´, Vandœuvre-le`s-Nancy, France Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling. Cancer Gene Therapy (2009) 16, 498–507; doi:10.1038/cgt.2008.100; published online 23 January 2009 Keywords: EGF receptor inhibitors; cetuximab; P53; PTEN; gene transfer Introduction number of patients whose tumors overexpress EGFRdo not respond to targeted therapy.6,7 In prostate cancer, The high frequency of overexpression of epidermal growth frequent gene mutations concern the p53 and phosphatase factor receptor (EGFR) in various cancers1 and its major and tensin homologue deleted on chromosome 10 (pten) function in tumorigenesis have suggested that inhibition of tumor suppressor genes. P53 is a transcription factor that EGFRstimulation and inhibition of downstream signal- regulates the cell-cycle arrest, DNA repair and apoptosis. ing pathways could represent new therapeutic approaches In fact, p53 is probably the most frequently mutated gene in cancer treatment and have opened the way of targeted in human cancer. Loss of p53 contributes to tumor therapies. Different concepts have been proposed for progression, therapeutic resistance and is often associated inhibition of EGFRactivation including monoclonal with a poor prognosis. The rate of mutations in prostate antibodies and tyrosine kinase inhibitors. Cetuximab is a cancer in the p53 tumor suppressor gene varied among chimeric monoclonal antibody developed for therapeutic tumors of different aggressiveness, ranging from 5% for purposes, which binds to EGFRwith high affinity and patients with clinically localized disease to 62% in 8 abrogates ligand-induced EGFRphosphorylation. 2 In metastatic disease. P53 may also affect the signaling preclinical models, EGFRinhibition by monoclonal pathways that are involved in cell growth and transforma- antibody or by the use of tyrosine kinase inhibitors has tion. P53 reintroduction has been shown to significantly been associated with decreased cell proliferation, increased produce greater apoptosis and tumor growth suppression 9 apoptosis and decreased angiogenesis.3–5 However, a large than any other drug alone. Recent data showed that sensitivity to gefitinib is affected by P53 mutation and that P53 inhibition induces a significant decrease in apoptosis Correspondence: Professor J-L Merlin, Unite´ de Biologie des 10 Tumeurs, EA3452 Nancy Universite´ , Centre Alexis Vautrin, 6 induction by gefitinib. Previous study already demon- Avenue de Bourgogne, 54511 Vandœuvre-le` s-Nancy cedex, France. strated that cetuximab inhibited the growth of wild-type E-mail: [email protected] p53 but not of mutated p53 cancer cells and hypothesized Received 8 April 2008; revised 25 October 2008; accepted 26 that resistance to cetuximab could relate to p53 11 November 2008; published online 23 January 2009 mutation. In addition, recent study showed that P53, PTEN and cetuximab sensitivity S Bouali et al 499 induction of P53 resulted in decreased in phosphorylated inactivated fetal calf serum (Dutscher, Brumath, France) AKT activity in cell line, which did not express PTEN and 1% glutamine. Culture media and additives were protein, thus highlighting that p53 was involved in from Life Technologies (Eragny, France). regulation of survival PI3K/AKT pathway in epithelial 12 tumors, and independently of PTEN expression. It was Gene transfer reported that activation of p53-dependent downstream Linear polyethylenimine (PEI) was kindly provided by targets BAX with concomitant increase of MAPKinase Polyplus-transfection (Illkirch, France). pCMV-Luc 13 (MAPK) pathways leading to apoptosis. Altogether, (Dr P Erbacher, Polyplus-transfection), pNCNeo (Dr these data indicated that the relationship between P53, JM Heard, Institut Pasteur, Paris, France) and pPTEN PI3K/AKT and MAPK signaling pathways offers novel (Dr K Yamada, NIH, Bethesda, MD) plasmids, encoding valuable insights into the resistance to targeted therapy. luciferase, p53 and pten genes, respectively, were purified 14 Pten was identified in 1997 as tumor suppressor and from Escherichia coli using Marligen maxiprep columns alteration of pten gene has been shown to promote (Life Technologies). 15 prostate cancer progression. PTEN antagonizes the Gene transfer was performed as already published20 action of PI3K and negatively controls the downstream with slight modifications. Briefly, the cells were seeded at 16 pathway by inhibiting the phosphorylation of AKT. the density of 5 Â 104 mlÀ1 in six-well plates 48 h before Consequently, the loss of PTEN expression leads to an transfection to reach 60–70% confluence at the time of 17 elevated activation of AKT. A recent study investigating transfection. pCMV-Luc or pNCNeo or pPTEN plasmids protein expression demonstrated that PTEN has a (5 mgmlÀ1) and corresponding amount of PEI were fundamental function in predicting the response to separately prepared and the resulting mixture was added cetuximab against EGFRin metastatic colorectal to the cells. Twenty-four hours after transfection cells 18 cancer. A previous study showed that loss of PTEN were submitted to 100 mgmlÀ1 cetuximab (Merck Lipha expression was associated with resistance to gefitinib, and Sante, Lyon, France) and analyzed for cytotoxicity and that reintroduction of PTEN restored cell sensitivity of apoptotic induction. Pten and p53 co-transfer was gefitinib, thus suggesting that signaling pathway may achieved using 2.5 mgmlÀ1 of each plasmid. have a central function in the development of resistance to 19 EGFRinhibitors. In the present study, we used PC3 mRNA expression analysis human prostate carcinoma cell line harboring P53 Pten-mRNA and p53-mRNA expression was analyzed using and PTEN mutations. Our investigation was designed semiquantitative RT-PCR with b2-microglobulin (b2M1) as to evaluate whether p53 transfer could enhance PTEN- reference housekeeping gene as already reported.20,21 mediated growth inhibition and apoptosis induction to investigate the implication of PI3K/AKT and MAPK Western blot signaling proteins in response to cetuximab. Cells were exposed to epidermal growth factor (EGF, 100 ng mlÀ1) for 5 min (Roche Diagnostics, Penzberg, Germany). Western blots were carried out with total Materials and methods protein extracted from cells as already described using Cell lines monoclonal antibody directed against PTEN, pPTEN, PC3 cell line was obtained from the American Type P53, AKT, phosphorylated-AKT (pAKT), GSK3b, Culture Collection. The cells were cultured at 37 1C/5% pGSK3b, P70S6K, pP70S6K, ERK1/2, pERK1/2, BAX 22 CO2 in RPMI 1640, supplemented with 10% heat and tubulin (Table 1). Antibodies were used between Table 1 List of primary antibodies Proteins MW (kDa) Antibody Source Supplier Dilution EGFR 175 EGFR Rabbit Cell Signaling 1:1000 pEGFR 175 Phospho-EGFR (Tyr1068) Mouse Cell Signaling 1:1000 PTEN 54 PTEN Mouse Santa Cruz 1:500 pPTEN 54 Phospho-PTEN (Ser473) Rabbit Cell Signaling 1:1000 P53 53 P53 Rabbit Cell Signaling 1:1000 AKT 60 AKT Rabbit Cell Signaling 1:1000 pAKT 60 Phospho-AKT (Ser473) Rabbit Cell Signaling 1:1000 GSK3b 46 GSK Rabbit Cell Signaling 1:3000 pGSK3b 46 Phospho-GSK3b (Ser21/9) Rabbit Cell Signaling 1:2000 P70S6K 71 P70S6K Rabbit Cell Signaling 1:1000 pP70S6K 71 Phospho-P70S6K(Thr389) Rabbit Cell Signaling 1:1000 ERK1/2 42 ERK Rabbit Cell Signaling 1:2000 pERK1/2 42 Phospho-ERK (Thr202/Tyr204) Mouse Cell Signaling 1:1000 BAX 20 BAX Rabbit Dako 1:1000 Tubuline 27 Tubuline Rabbit Santa Cruz 1:500 Cancer Gene Therapy P53, PTEN and cetuximab sensitivity S Bouali et al 500 1:500 and
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