- Email: [email protected]
497: Activation of TRKA receptor by nerve growth factor induces shedding of P75 receptor related with progression of epithelial ovarian cancer
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 downstream RTKs, among which HER2, and its role in sustaining tumorigenic signals such as AKT activation, suggest that HER2 tumors may represent a model system suitable for testing the hypothesis that ATM may differently modulate tumor progression depending on the tumor environment. Material and Method: Lentivirus infection to genetically interfere ATM expression; immunoprecipitation and immunoblotting analysis; in vitro and in vivo tumorigenicity assay; immunohistochemistry (IHC). Results and Discussion: We found that ATM targeting significantly impairs HER2-dependent tumorigenicity in vitro. Moreover, xenograft experiments indicate that ATM may sustain HER2 tumorigenicity also in vivo. To ascertain whether ATM promotes in vivo HER2 dependent tumorigenicity we found also that ATM inhibitor KU55933 effectively decreased tumor multiplicity and size in transgenic MMTV-NeuT murine model. Moreover we identify ATM as a novel modulator of HER2 protein stability acting by promoting HER2 complex with HSP90 chaperone, therefore preventing its ubiquitination and degradation. As a consequence, ATM sustains AKT and MAPKs activation downstream HER2 and may modulate the response to therapeutic approaches, suggesting that the status of ATM activity may be informative for the treatment of HER2 positive tumors and for the prognosis. Importantly, patients (not treated with trastuzumab) bearing ATM-p positive/HER2 positive tumors have a shorter survival compared to ATM-p negative/HER2 positive tumors, supporting the idea that ATM enhances HER2 function. Conclusion: Our findings provide evidence for a tumorigenic potential of ATM revising the canonical role of ATM as pure tumor suppressor. No conflict of interest. 494 Prostaglandin E2 trans-activates the Colony-Stimulating Factor-1 (CSF-1) receptor and synergizes with CSF-1 in the induction of cell migration in macrophages via the mitogen-activated protein kinase ERK1/2 G. Digiacomo1 , M. Ziche2 , P. Dello Sbarba1 , S. Donnini2 , E. Rovida1 . University of Firenze, DSBSC, Firenze, Italy, 2 University of Siena, Dipartimento di Biotecnologie, Siena, Italy
1
Background: Prostaglandin E2 (PGE2) is a key mediator of immunopathology regulating multiple aspects of immune cells, inflammation and cancer. Besides the existence of four G-protein-coupled E-prostanoid receptors (EP1−4), the complexity of PGE2 signaling is further increased by the existence of a cross-talk between EP receptors and tyrosine kinase receptors (TKR). The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a TKR sustaining the survival, proliferation, and motility of monocytes/macrophages, essential component of innate immunity and cancer development. The aim of the study was to investigate on a possible cross-talk between PGE2- and CSF-1R-elicited signals. Material and Methods: All the experiments were performed with BAC-1.2F5 and RAW264.7 cells, two macrophage cell lines that physiologically express high amounts of CSF-1R protein. Kinases inhibition was performed either genetically (with siRNA) or pharmacologically with specific inhibitors. Western blotting and migration using modified Boyden chambers were performed at different time-points. Results: PGE2 induced a rapid CSF-1R phosphorylation. Genetic and pharmacological inhibition of CSF-1R reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that the CSF-1R plays a role in PGE2-mediated immuno-regulation. Furthermore, at low concentrations, PGE2 synergized with CSF-1 in inducing macrophage migration, an effect that was paralleled by a synergy on ERK1/2 phosphorylation. Accordingly, ERK1/2 inhibition completely blocked the migration induced by the combination CSF-1/PGE2. Conclusions: Our results indicate that PGE2 trans-activates CSF-1R and synergizes with its signaling at the level of ERK1/2 in promoting macrophage migration. No conflict of interest. 495 AKT and gefitinib resistance in mutant KRAS non-small cell lung cancers through mechanisms dependent of acetylation V. Jeannot1 , B. Busser1 , E. Brambilla1 , M. Wislez2 , B. Robin1 , J. Cadranel2 , J.L. Coll1 , A. Hurbin1 . 1 INSERM U823, IAB, Grenoble Cedex 09, France, 2 AP-HP Tenon Hospital, Pneumology Dept, Paris, France Background: Epidermal growth factor receptor (EGFR) is frequently overexpressed in non-small cell lung cancers (NSCLC) and is associated with poor prognosis. While therapies targeting the tyrosine kinase activity of EGFR (EGFR-TKI, such as gefitinib) are highly effective for the treatment of EGFR mutated NSCLC, limited response rates are observed in EGFR wildtype NSCLC. We previously reported that amphiregulin and insulin-like growth factor-1 receptor (IGF1R) overexpression lead to gefitinib resistance in mutant KRAS adenocarcinoma, though Ku70 acetylation inhibition, that enhances the BAX/Ku70 interaction and prevents apoptosis. Here we characterized the intracellular pathways leading to EGFR-TKI resistance, in order to define therapeutic targets.
S119
Material and Method: We analyzed the activation of intracellular pathways, phosphatidyl inositol-3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK), in lung tumors, including patients treated with gefitinib (phase II clinical trial IFCT0401). We explored the impact of inhibition of these intracellular pathways in mutant KRAS cells. Results: The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib. The inhibition of amphiregulin or IGF1R decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. PI3K/AKT inhibition combined with gefitinib restored apoptosis, through Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which released BAX from Ku70, inhibited AKT signaling and induced gefitinib-dependent apoptosis. Conclusion: The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in mutant KRAS adenocarcinoma, by a deacetylasedependent mechanism. The PI3K/AKT pathway induces survival of wild-type EGFR NSCLC with KRAS mutation, suggesting new therapeutic target for treating this subset of lung cancer patients, who have a poor prognosis. No conflict of interest. 496 Nuclear EGFR controls the expression of the ARF tumor suppressor D. Dayde1 , P. Ozenne1 , P. Perron1 , C. Barrial1 , B. Eymin1 , S. Gazzeri1 . INSERM U823, Grenoble Cedex 09, France
1
Background: The Epidermal Growth Factor Receptor (EGFR) is a driver oncogene involved in lung cancer development. Activation of EGFR by its ligands induces several signaling cascades stimulating proliferation and cell survival. Beside this classic signalisation initiated by EGFR at the plasma membrane, it is now recognized that EGFR can also shuttle from the cell surface to the nucleus and acts as a co-transcription factor for genes mainly involved in cell proliferation. The tumor suppressor gene ARF inhibits the development of lung cancer. Our recent data demonstrate that activated mutant EGFR counteracts the pro-apoptotic function of ARF by downregulating its expression to promote lung carcinogenesis [1]. The aim of this study was to investigate the mechanisms involved in ARF control by EGFR. Material and Methods: ARF expression was studied by RT/QCR and western blotting in various lung tumor cell lines with distinct EGFR status (H1719 WT EGFR, HCC827 and H1975 mutant EGFR). Nuclear expression of EGFR was analyzed by western blotting following subcellular fractionation. siRNA against importinb1 and pharmacological inhibitors and were used to control nuclear trafficking of EGFR and to study the role of canonic EGFR signalling pathways (MEK/ERK, PI3K/AKT, STAT3, SRC) in this context. Transcriptional control of ARF by nuclear EGFR was confirmed by Chromatin immunoprecipitation. Results: In various lung tumor cell lines we demonstrate that activation of EGFR by ligand binding inhibits in a dose and time-dependent manner the expression the of ARF transcripts. Blocking nuclear trafficking partially prevents the ability of EGFR to repress ARF mRNA following EGF treatment. Using ChIP we provide evidence that activated EGFR binds the promoter of ARF confirming the control of ARF transcription by nuclear EGFR. Finally, we provide evidence that inhibition of PI3K/AKT signalling pathway prevents the nuclear trafficking of EGFR and further repression of ARF transcription. Conclusions: These data identify a new transcriptional repressor function for nuclear EGFR and suggest that nuclear signalisation of EGFR might play a role in lung carcinogenesis through the repression of ARF expression. Reference(s) [1] Ozenne P et al, Oncogene, 2013. No conflict of interest. 497 Activation of TRKA receptor by nerve growth factor induces shedding of P75 receptor related with progression of epithelial ovarian cancer C. Romero1 , C. Vallejos2 , F. Gabler3 , A. Selman4 , M. Vega5 . 1 Hospital Clinico Universidad de Chile, Obstetricia y Ginecolog´ıa Laboratorio de ´ ACCDiS, Santiago, Chile, Endocrinolog´ıa y Biolog´ıa de la Reproduccion 2 Hospital Clinico Universidad de Chile, Laboratorio de Endocrinolog´ıa ´ Santiago, Chile, 3 Facultad de Medicina y Biolog´ıa de la Reproduccion, ´ Universidad de Chile, Departamento de Anatom´ıa Patologica, Santiago, Chile, 4 Hospital Cl´ınico Universidad de Chile, Departamento Obstetricia 5 y Ginecolog´ıa, Santiago, Chile, Hospital Cl´ınico Universidad de Chile, Departamento Obstetricia y Ginecolog´ıa Laboratorio de Endocrinolog´ıa y ´ Santiago, Chile Biolog´ıa de la Reproduccion, Introduction: Nerve growth factor (NGF) and its high affinity receptor TRKA are highly expressed in epithelial ovarian cancer (EOC) and also involved in proliferation and angiogenesis. We found an increase of ADAM17 in EOC and a positive correlation with TRKA. In prostate and breast cancer, TRKA is also increased; but P75, low affinity receptor of NGF is decreased, suggesting a negative correlation between P75 expression and cancer malignancy. In
S120
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
other tissues, TRKA activation by NGF induces a shedding of P75 receptor by ADAM17, finding two forms: full length receptor (P75FL ) and intracellular domain with trans-membrane domain (P75CTF ). The aim of this work was to evaluate whether P75 mRNA and protein levels change with the progression of epithelial ovarian cancer and also if the activation of TRKA by NGF induces the P75 shedding by ADAM17 in two cell lines: human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780). Material and Methods: Fifty human ovarian tissues: inactive normal ovaries, ovarian tumors and epithelial ovarian cancer (well differentiated, moderate differentiated, and poorly differentiated) were obtained from Obstetric and Gynecology Department, Clinical Hospital University of Chile, with signed consent approved by Institutional Ethic Committee. In all samples P75 mRNA levels were measured by qPCR and the P75 protein levels were detected by IHC with antibody that recognizes the extracellular domain (p75FL ). HOSE and A2780 cells were stimulated with NGF; TRKA inhibitors (GW441756); ADAM17 inhibitors (Tapi0) and NGF plus the inhibitors. The P75 mRNA levels were measured by qPCR and the protein levels by western-blot with two different antibodies; one recognizes P75FL and the other one P75CTF . Results and Discussion: The detection of P75FL protein In poorly differentiated EOC was lower compared with moderated EOC as well as well differentiated EOC (p < 0.01); the mRNA levels of P75 were lower in poorly differentiated EOC compare with well differentiated EOC (p < 0.05). In A2780 cells, P75FL protein levels were 51% lower than in HOSE cells in control conditions and when these cell lines were stimulated with NGF, P75FL decreased 77.6% in HOSE cells and 52% in A2780 cells compared with the respective controls. When we used TRKA and ADAM17 inhibitors alone or with NGF, P75FL protein levels were similar to control in both cell lines; the same results were found with Anti-NGF alone or plus NGF. In control condition, the P75CTF protein levels in A2780 cells were 64.7% higher than in HOSE cells and only in HOSE cells it was observed a significant increase 118% of P75CTF protein levels with NGF (p < 0.01). The others conditions studied no changes were found with respect to controls. Conclusion: These results suggest that P75FL decreases with EOC progression probably due to the activation of TRKA by NGF, by inducing a shedding of P75 receptor by ADAM17 and increasing P75CTF . This work was funded by FONDECYT 1110372 to CR. No conflict of interest. 498 Two distinct antitumor pathways activated by transfected poly(I:C) in androgen-independent prostate cancer cells S. Palchetti1 , D. Starace1 , P. De Cesaris1 , A. Filippini1 , E. Ziparo1 , A. Riccioli1 . 1 University of Rome “Sapienza”, DAHFMO − Section of Histology and Medical Embryology, Roma, Italy Introduction: Prostate cancer (PCa) represents the second leading cause of cancer death in men and develops as a result of the accumulation of genetic and epigenetic alterations. Data from literature suggest that new therapeutic targets are emerging and in particular, it is known that the activation of Tolllike Receptors 3 (TLR3) expressed by cancer cells has a pro-apoptotic and thus anti-tumoral effect in different tumors (Cheng & Xu, 2010). We previously demonstrated that the synthetic analogue of dsRNA poly(I:C), specific TLR3ligand, induces apoptosis in the androgen-dependent prostate cancer cell line LNCaP in a TLR3-dependent fashion, whereas a weaker apoptotic effect is observed in more aggressive and androgen-independent prostate cancer cell lines PC3 (Paone et al., 2008) and DU-145 (Galli et al., 2013). In this regard, we have recently demonstrated that the encapsulation of poly(I:C) with three different formulations of cationic liposomes was up to 10 times more efficient than the free drug in eliminating both PC3 and DU145 cells (Palchetti et al., 2013). These data suggest that transfected poly(I:C) could raise apoptotic rate by stimulating cytosolic dsRNA receptors. In the present paper we analyzed the receptors and signalling pathways involved in apoptosis induced by poly(I:C) transfected by lipofectamine (the most common transfection agent) compared with free poly(I:C) in PC3 and DU145 cells. Material and Method: We evaluated cell viability by MTT assay and apoptosis by cell cycle analysis by FACS and caspase activity. SiRNA approach and Western Blot analysis were performed to determine the receptors and signal transduction molecules involved in transfected poly(I:C)-induced effects. Results: Poly(I:C) transfected by lipofectamine [in-poly(I:C)] inhibits cell viability in PC3 and DU145 cells in a dose dependent manner with the highest efficiency at 2 mg/ml of poly(I:C) compared to twelve-fold higher poly(I:C) concentration (25 mg/ml) and induces caspase 8 and 9-dependent apoptosis. By using genetic inhibition of different poly(I:C) receptors we demonstrated the crucial role of TLR3 and Src in in-poly(I:C) induced apoptosis. On the other hand, we show that IRF3-mediated signaling causes the upregulation of TLR3, cytosolic receptors (RLH) and interferon-beta expression. Our data highlight the multiple signaling triggered by in-poly(I:C) leading to antitumor responses. Conclusion: We can conclude that the treatment of PC3 and DU145 cells with in-poly(I:C) activates two distinct anti-tumor pathways: one mediated by TLR3, dependent on Src, leading to a remarkable apoptosis and the other
one mediated by RLH, dependent on IRF-3, leading to their up-regulation and interferon-beta expression. No conflict of interest. 499 The role of Hedgehog signaling pathway in the regulation of SOX18 gene expression in cervical carcinoma cell line I. Petrovic1 , M. Milivojevic1 , M. Mojsin1 , D. Drakulic1 , N. Kovacevic Grujicic1 , V. Topalovic1 , S. Davidovic1 , M. Stevanovic1 . 1 Institute of Molecular Genetics and Genetic Engineering University of Belgrade, Laboratory for Human Molecular Genetics, Belgrade, Serbia Background: Many genes that were first identified as key genes involved in regulation of embryogenesis, have been found to play essential roles in cancer development. SOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development. In addition, it has been reported that SOX18 affects the growth of cancer cells in vitro. We have investigated whether activation of Hh-Gli signaling pathway affects transcriptional regulation of SOX18 gene expression and consequently viability and proliferation of cervical carcinoma cells in vitro. Materials and Methods: As a model system for cervical carcinoma we have used HeLa cell line. The role of Gli transcription factors, as final effectors of Hh-Gli pathway, in the regulation of SOX18 gene expression was investigated by transient co-transfections followed by functional analyses of SOX18 promoter activity, and analysis of endogenous SOX18 expression by qRT-PCR and Western blot. In vitro binding of Gli transcription factors to predicted binding sites within SOX18 promoter was investigated by EMSA. The effect of Hh-Gli-pathway activation or inhibition on cell proliferation and viability was investigated by MTT test and the effect of these modulations on SOX18 expression was analyzed by qRT-PCR and Western blot. Results: We have shown that transcription factors Gli1 and Gli2 are potent activators of SOX18 promoter activity and SOX18 expression in HeLa cells, while Gli3 had no significant effect. We have shown in vitro binding of Gli1 to two out of seven predicted binding sites within SOX18 promoter. In order to investigate whether this Gli associated activation of SOX18 expression is linked to canonical Hedgehog signaling we have included in our analysis modulation of Hh-Gli signaling by employing specific inhibitors and activators. Inhibition of Hh-Gli signaling pathway reduced proliferation of HeLa cells and also impaired the cells viability. Also, inhibition of pathway led to reduction of SOX18 expression. On the other hand, activation of pathway showed opposite results. These results showed that SOX18 expression is, in part, regulated by Hh-Gli signaling pathway in HeLa cells. Conclusions: SOX18 gene is novel target of Hh-Gli signaling pathway. This pro-angiogenic transcription factor could have an important role in Hh-activated proliferation of cervical carcinoma cells. No conflict of interest. 500 Analysis of the metastasis/tumor-suppressing effects of SASH1 U. Nitsche1 , E. Lichtenegger1 , S. Leis1 , B. Heckl1 , H. Weidmann2 , E. Nino2 , K.P. Janssen1 . 1 Klinikum rechts der Isar, Chirurgische Klinik und Poliklinik, ´ Munchen, ¨ Germany, 2 Faculte´ de Medecine Pierre et Marie Curie, Inserm UMR 1166, Paris, France Background: SASH1 (SAM- and SH3-domain containing 1) is a candidate tumor suppressor in colon and breast cancer. Recently, we identified a prognostic relevance of reduced SASH1 expression for metastasis development in patients with colorectal cancer. However, the biological role of SASH1 is largely unclear. SASH1 shows a broad expression pattern, it has nuclear and cytosolic localisation, interacts with the cytoskeleton, induces actin polymerization and cell–matrix-adhesion, potentially leading to reduced invasiveness of tumor cells. Here, we report on the discovery of two putative downstream targets of SASH1, providing a possible explanation for its high prognostic relevance. Moreover, we propose a new gene-trap mouse model to study the role of SASH1 in vivo. Material and Methods: Microarray analysis of the colorectal cancer cell line SW480, with stable alteration of SASH1 expression levels, was performed to identify downstream targets. Putative target genes and their signalling interactions with SASH1 were further validated by qRT-PCR in vitro. Finally, human tumor specimens and a recently established SASH1 genetrap-mouse model, based on the integration of a beta-GEO gene-trap cassette in the murine SASH1 gene locus, were analyzed for additional validation. Results: Transcriptome analysis of SW480 cells identified a massive upregulation of transcripts after stable SASH1 knockdown, whereas increased expression of SASH1 resulted in minor changes. Therefore, SASH1 seems to have an inhibitory effect on gene transcription. Among the transcripts negatively regulated by SASH1, we identified MACC1 (Metastasis associated in colon cancer) and Cyclin D3 (CCND3). MACC1 has been described to promote tumor growth by activation of the HGF/cMET pathway, whereas Cyclin D3 leads to cell cycle progression. Further cell line experiments confirmed SASH1 knockdown to lead to upregulation of expression levels of MACC1, as well as Cyclin D3. These results were confirmed by analysis of the
1
Background: Prostaglandin E2 (PGE2) is a key mediator of immunopathology regulating multiple aspects of immune cells, inflammation and cancer. Besides the existence of four G-protein-coupled E-prostanoid receptors (EP1−4), the complexity of PGE2 signaling is further increased by the existence of a cross-talk between EP receptors and tyrosine kinase receptors (TKR). The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a TKR sustaining the survival, proliferation, and motility of monocytes/macrophages, essential component of innate immunity and cancer development. The aim of the study was to investigate on a possible cross-talk between PGE2- and CSF-1R-elicited signals. Material and Methods: All the experiments were performed with BAC-1.2F5 and RAW264.7 cells, two macrophage cell lines that physiologically express high amounts of CSF-1R protein. Kinases inhibition was performed either genetically (with siRNA) or pharmacologically with specific inhibitors. Western blotting and migration using modified Boyden chambers were performed at different time-points. Results: PGE2 induced a rapid CSF-1R phosphorylation. Genetic and pharmacological inhibition of CSF-1R reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that the CSF-1R plays a role in PGE2-mediated immuno-regulation. Furthermore, at low concentrations, PGE2 synergized with CSF-1 in inducing macrophage migration, an effect that was paralleled by a synergy on ERK1/2 phosphorylation. Accordingly, ERK1/2 inhibition completely blocked the migration induced by the combination CSF-1/PGE2. Conclusions: Our results indicate that PGE2 trans-activates CSF-1R and synergizes with its signaling at the level of ERK1/2 in promoting macrophage migration. No conflict of interest. 495 AKT and gefitinib resistance in mutant KRAS non-small cell lung cancers through mechanisms dependent of acetylation V. Jeannot1 , B. Busser1 , E. Brambilla1 , M. Wislez2 , B. Robin1 , J. Cadranel2 , J.L. Coll1 , A. Hurbin1 . 1 INSERM U823, IAB, Grenoble Cedex 09, France, 2 AP-HP Tenon Hospital, Pneumology Dept, Paris, France Background: Epidermal growth factor receptor (EGFR) is frequently overexpressed in non-small cell lung cancers (NSCLC) and is associated with poor prognosis. While therapies targeting the tyrosine kinase activity of EGFR (EGFR-TKI, such as gefitinib) are highly effective for the treatment of EGFR mutated NSCLC, limited response rates are observed in EGFR wildtype NSCLC. We previously reported that amphiregulin and insulin-like growth factor-1 receptor (IGF1R) overexpression lead to gefitinib resistance in mutant KRAS adenocarcinoma, though Ku70 acetylation inhibition, that enhances the BAX/Ku70 interaction and prevents apoptosis. Here we characterized the intracellular pathways leading to EGFR-TKI resistance, in order to define therapeutic targets.
S119
Material and Method: We analyzed the activation of intracellular pathways, phosphatidyl inositol-3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK), in lung tumors, including patients treated with gefitinib (phase II clinical trial IFCT0401). We explored the impact of inhibition of these intracellular pathways in mutant KRAS cells. Results: The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib. The inhibition of amphiregulin or IGF1R decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. PI3K/AKT inhibition combined with gefitinib restored apoptosis, through Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which released BAX from Ku70, inhibited AKT signaling and induced gefitinib-dependent apoptosis. Conclusion: The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in mutant KRAS adenocarcinoma, by a deacetylasedependent mechanism. The PI3K/AKT pathway induces survival of wild-type EGFR NSCLC with KRAS mutation, suggesting new therapeutic target for treating this subset of lung cancer patients, who have a poor prognosis. No conflict of interest. 496 Nuclear EGFR controls the expression of the ARF tumor suppressor D. Dayde1 , P. Ozenne1 , P. Perron1 , C. Barrial1 , B. Eymin1 , S. Gazzeri1 . INSERM U823, Grenoble Cedex 09, France
1
Background: The Epidermal Growth Factor Receptor (EGFR) is a driver oncogene involved in lung cancer development. Activation of EGFR by its ligands induces several signaling cascades stimulating proliferation and cell survival. Beside this classic signalisation initiated by EGFR at the plasma membrane, it is now recognized that EGFR can also shuttle from the cell surface to the nucleus and acts as a co-transcription factor for genes mainly involved in cell proliferation. The tumor suppressor gene ARF inhibits the development of lung cancer. Our recent data demonstrate that activated mutant EGFR counteracts the pro-apoptotic function of ARF by downregulating its expression to promote lung carcinogenesis [1]. The aim of this study was to investigate the mechanisms involved in ARF control by EGFR. Material and Methods: ARF expression was studied by RT/QCR and western blotting in various lung tumor cell lines with distinct EGFR status (H1719 WT EGFR, HCC827 and H1975 mutant EGFR). Nuclear expression of EGFR was analyzed by western blotting following subcellular fractionation. siRNA against importinb1 and pharmacological inhibitors and were used to control nuclear trafficking of EGFR and to study the role of canonic EGFR signalling pathways (MEK/ERK, PI3K/AKT, STAT3, SRC) in this context. Transcriptional control of ARF by nuclear EGFR was confirmed by Chromatin immunoprecipitation. Results: In various lung tumor cell lines we demonstrate that activation of EGFR by ligand binding inhibits in a dose and time-dependent manner the expression the of ARF transcripts. Blocking nuclear trafficking partially prevents the ability of EGFR to repress ARF mRNA following EGF treatment. Using ChIP we provide evidence that activated EGFR binds the promoter of ARF confirming the control of ARF transcription by nuclear EGFR. Finally, we provide evidence that inhibition of PI3K/AKT signalling pathway prevents the nuclear trafficking of EGFR and further repression of ARF transcription. Conclusions: These data identify a new transcriptional repressor function for nuclear EGFR and suggest that nuclear signalisation of EGFR might play a role in lung carcinogenesis through the repression of ARF expression. Reference(s) [1] Ozenne P et al, Oncogene, 2013. No conflict of interest. 497 Activation of TRKA receptor by nerve growth factor induces shedding of P75 receptor related with progression of epithelial ovarian cancer C. Romero1 , C. Vallejos2 , F. Gabler3 , A. Selman4 , M. Vega5 . 1 Hospital Clinico Universidad de Chile, Obstetricia y Ginecolog´ıa Laboratorio de ´ ACCDiS, Santiago, Chile, Endocrinolog´ıa y Biolog´ıa de la Reproduccion 2 Hospital Clinico Universidad de Chile, Laboratorio de Endocrinolog´ıa ´ Santiago, Chile, 3 Facultad de Medicina y Biolog´ıa de la Reproduccion, ´ Universidad de Chile, Departamento de Anatom´ıa Patologica, Santiago, Chile, 4 Hospital Cl´ınico Universidad de Chile, Departamento Obstetricia 5 y Ginecolog´ıa, Santiago, Chile, Hospital Cl´ınico Universidad de Chile, Departamento Obstetricia y Ginecolog´ıa Laboratorio de Endocrinolog´ıa y ´ Santiago, Chile Biolog´ıa de la Reproduccion, Introduction: Nerve growth factor (NGF) and its high affinity receptor TRKA are highly expressed in epithelial ovarian cancer (EOC) and also involved in proliferation and angiogenesis. We found an increase of ADAM17 in EOC and a positive correlation with TRKA. In prostate and breast cancer, TRKA is also increased; but P75, low affinity receptor of NGF is decreased, suggesting a negative correlation between P75 expression and cancer malignancy. In
S120
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
other tissues, TRKA activation by NGF induces a shedding of P75 receptor by ADAM17, finding two forms: full length receptor (P75FL ) and intracellular domain with trans-membrane domain (P75CTF ). The aim of this work was to evaluate whether P75 mRNA and protein levels change with the progression of epithelial ovarian cancer and also if the activation of TRKA by NGF induces the P75 shedding by ADAM17 in two cell lines: human ovarian surface epithelial (HOSE) and epithelial ovarian cancer (A2780). Material and Methods: Fifty human ovarian tissues: inactive normal ovaries, ovarian tumors and epithelial ovarian cancer (well differentiated, moderate differentiated, and poorly differentiated) were obtained from Obstetric and Gynecology Department, Clinical Hospital University of Chile, with signed consent approved by Institutional Ethic Committee. In all samples P75 mRNA levels were measured by qPCR and the P75 protein levels were detected by IHC with antibody that recognizes the extracellular domain (p75FL ). HOSE and A2780 cells were stimulated with NGF; TRKA inhibitors (GW441756); ADAM17 inhibitors (Tapi0) and NGF plus the inhibitors. The P75 mRNA levels were measured by qPCR and the protein levels by western-blot with two different antibodies; one recognizes P75FL and the other one P75CTF . Results and Discussion: The detection of P75FL protein In poorly differentiated EOC was lower compared with moderated EOC as well as well differentiated EOC (p < 0.01); the mRNA levels of P75 were lower in poorly differentiated EOC compare with well differentiated EOC (p < 0.05). In A2780 cells, P75FL protein levels were 51% lower than in HOSE cells in control conditions and when these cell lines were stimulated with NGF, P75FL decreased 77.6% in HOSE cells and 52% in A2780 cells compared with the respective controls. When we used TRKA and ADAM17 inhibitors alone or with NGF, P75FL protein levels were similar to control in both cell lines; the same results were found with Anti-NGF alone or plus NGF. In control condition, the P75CTF protein levels in A2780 cells were 64.7% higher than in HOSE cells and only in HOSE cells it was observed a significant increase 118% of P75CTF protein levels with NGF (p < 0.01). The others conditions studied no changes were found with respect to controls. Conclusion: These results suggest that P75FL decreases with EOC progression probably due to the activation of TRKA by NGF, by inducing a shedding of P75 receptor by ADAM17 and increasing P75CTF . This work was funded by FONDECYT 1110372 to CR. No conflict of interest. 498 Two distinct antitumor pathways activated by transfected poly(I:C) in androgen-independent prostate cancer cells S. Palchetti1 , D. Starace1 , P. De Cesaris1 , A. Filippini1 , E. Ziparo1 , A. Riccioli1 . 1 University of Rome “Sapienza”, DAHFMO − Section of Histology and Medical Embryology, Roma, Italy Introduction: Prostate cancer (PCa) represents the second leading cause of cancer death in men and develops as a result of the accumulation of genetic and epigenetic alterations. Data from literature suggest that new therapeutic targets are emerging and in particular, it is known that the activation of Tolllike Receptors 3 (TLR3) expressed by cancer cells has a pro-apoptotic and thus anti-tumoral effect in different tumors (Cheng & Xu, 2010). We previously demonstrated that the synthetic analogue of dsRNA poly(I:C), specific TLR3ligand, induces apoptosis in the androgen-dependent prostate cancer cell line LNCaP in a TLR3-dependent fashion, whereas a weaker apoptotic effect is observed in more aggressive and androgen-independent prostate cancer cell lines PC3 (Paone et al., 2008) and DU-145 (Galli et al., 2013). In this regard, we have recently demonstrated that the encapsulation of poly(I:C) with three different formulations of cationic liposomes was up to 10 times more efficient than the free drug in eliminating both PC3 and DU145 cells (Palchetti et al., 2013). These data suggest that transfected poly(I:C) could raise apoptotic rate by stimulating cytosolic dsRNA receptors. In the present paper we analyzed the receptors and signalling pathways involved in apoptosis induced by poly(I:C) transfected by lipofectamine (the most common transfection agent) compared with free poly(I:C) in PC3 and DU145 cells. Material and Method: We evaluated cell viability by MTT assay and apoptosis by cell cycle analysis by FACS and caspase activity. SiRNA approach and Western Blot analysis were performed to determine the receptors and signal transduction molecules involved in transfected poly(I:C)-induced effects. Results: Poly(I:C) transfected by lipofectamine [in-poly(I:C)] inhibits cell viability in PC3 and DU145 cells in a dose dependent manner with the highest efficiency at 2 mg/ml of poly(I:C) compared to twelve-fold higher poly(I:C) concentration (25 mg/ml) and induces caspase 8 and 9-dependent apoptosis. By using genetic inhibition of different poly(I:C) receptors we demonstrated the crucial role of TLR3 and Src in in-poly(I:C) induced apoptosis. On the other hand, we show that IRF3-mediated signaling causes the upregulation of TLR3, cytosolic receptors (RLH) and interferon-beta expression. Our data highlight the multiple signaling triggered by in-poly(I:C) leading to antitumor responses. Conclusion: We can conclude that the treatment of PC3 and DU145 cells with in-poly(I:C) activates two distinct anti-tumor pathways: one mediated by TLR3, dependent on Src, leading to a remarkable apoptosis and the other
one mediated by RLH, dependent on IRF-3, leading to their up-regulation and interferon-beta expression. No conflict of interest. 499 The role of Hedgehog signaling pathway in the regulation of SOX18 gene expression in cervical carcinoma cell line I. Petrovic1 , M. Milivojevic1 , M. Mojsin1 , D. Drakulic1 , N. Kovacevic Grujicic1 , V. Topalovic1 , S. Davidovic1 , M. Stevanovic1 . 1 Institute of Molecular Genetics and Genetic Engineering University of Belgrade, Laboratory for Human Molecular Genetics, Belgrade, Serbia Background: Many genes that were first identified as key genes involved in regulation of embryogenesis, have been found to play essential roles in cancer development. SOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development. In addition, it has been reported that SOX18 affects the growth of cancer cells in vitro. We have investigated whether activation of Hh-Gli signaling pathway affects transcriptional regulation of SOX18 gene expression and consequently viability and proliferation of cervical carcinoma cells in vitro. Materials and Methods: As a model system for cervical carcinoma we have used HeLa cell line. The role of Gli transcription factors, as final effectors of Hh-Gli pathway, in the regulation of SOX18 gene expression was investigated by transient co-transfections followed by functional analyses of SOX18 promoter activity, and analysis of endogenous SOX18 expression by qRT-PCR and Western blot. In vitro binding of Gli transcription factors to predicted binding sites within SOX18 promoter was investigated by EMSA. The effect of Hh-Gli-pathway activation or inhibition on cell proliferation and viability was investigated by MTT test and the effect of these modulations on SOX18 expression was analyzed by qRT-PCR and Western blot. Results: We have shown that transcription factors Gli1 and Gli2 are potent activators of SOX18 promoter activity and SOX18 expression in HeLa cells, while Gli3 had no significant effect. We have shown in vitro binding of Gli1 to two out of seven predicted binding sites within SOX18 promoter. In order to investigate whether this Gli associated activation of SOX18 expression is linked to canonical Hedgehog signaling we have included in our analysis modulation of Hh-Gli signaling by employing specific inhibitors and activators. Inhibition of Hh-Gli signaling pathway reduced proliferation of HeLa cells and also impaired the cells viability. Also, inhibition of pathway led to reduction of SOX18 expression. On the other hand, activation of pathway showed opposite results. These results showed that SOX18 expression is, in part, regulated by Hh-Gli signaling pathway in HeLa cells. Conclusions: SOX18 gene is novel target of Hh-Gli signaling pathway. This pro-angiogenic transcription factor could have an important role in Hh-activated proliferation of cervical carcinoma cells. No conflict of interest. 500 Analysis of the metastasis/tumor-suppressing effects of SASH1 U. Nitsche1 , E. Lichtenegger1 , S. Leis1 , B. Heckl1 , H. Weidmann2 , E. Nino2 , K.P. Janssen1 . 1 Klinikum rechts der Isar, Chirurgische Klinik und Poliklinik, ´ Munchen, ¨ Germany, 2 Faculte´ de Medecine Pierre et Marie Curie, Inserm UMR 1166, Paris, France Background: SASH1 (SAM- and SH3-domain containing 1) is a candidate tumor suppressor in colon and breast cancer. Recently, we identified a prognostic relevance of reduced SASH1 expression for metastasis development in patients with colorectal cancer. However, the biological role of SASH1 is largely unclear. SASH1 shows a broad expression pattern, it has nuclear and cytosolic localisation, interacts with the cytoskeleton, induces actin polymerization and cell–matrix-adhesion, potentially leading to reduced invasiveness of tumor cells. Here, we report on the discovery of two putative downstream targets of SASH1, providing a possible explanation for its high prognostic relevance. Moreover, we propose a new gene-trap mouse model to study the role of SASH1 in vivo. Material and Methods: Microarray analysis of the colorectal cancer cell line SW480, with stable alteration of SASH1 expression levels, was performed to identify downstream targets. Putative target genes and their signalling interactions with SASH1 were further validated by qRT-PCR in vitro. Finally, human tumor specimens and a recently established SASH1 genetrap-mouse model, based on the integration of a beta-GEO gene-trap cassette in the murine SASH1 gene locus, were analyzed for additional validation. Results: Transcriptome analysis of SW480 cells identified a massive upregulation of transcripts after stable SASH1 knockdown, whereas increased expression of SASH1 resulted in minor changes. Therefore, SASH1 seems to have an inhibitory effect on gene transcription. Among the transcripts negatively regulated by SASH1, we identified MACC1 (Metastasis associated in colon cancer) and Cyclin D3 (CCND3). MACC1 has been described to promote tumor growth by activation of the HGF/cMET pathway, whereas Cyclin D3 leads to cell cycle progression. Further cell line experiments confirmed SASH1 knockdown to lead to upregulation of expression levels of MACC1, as well as Cyclin D3. These results were confirmed by analysis of the