- Email: [email protected]
504: Functional proteomic analysis of leukaemogenic protein tyrosine kinase targets
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 murine SASH1 gene-trap model. Moreover, mice carrying the gene-trap allele spontaneously developed solid tumors and had significantly reduced overall survival. Conclusions: Taken together, MACC1 and cyclin D3 represent tumor promoting factors negatively controlled by the upstream regulator SASH1. These findings provide an explanation for the high prognostic relevance of SASH1 in colorectal cancer, which had been previously found in clinical patient samples. No conflict of interest. 501 The prognostic value of b-catenin in anal cancer H. Jacob1 , O. Dahl1 , M. Myklebust1 . 1 Department of Clinical Science, University of Bergen Haukeland Hospital University, Bergen, Norway Background: Anal cancer is an uncommon malignancy whose incidence has risen markedly in several parts of the world. About 80% of all anal cancers are squamous cell carcinoma (SCC) and the standard treatment is the combination of radiotherapy and chemotherapy. Prognosis of patients varies and knowledge of clinical prognostic factors is critical for information about patient’s outcome. b-catenin, a key component of the Wnt (wingless) signaling pathway, is involved in regulation of E-cadherin mediated cell adhesion. In presence of Wnt ligand, b-catenin accumulates in the nucleus and binds to transcription factors. It is also associated with the adenomatous polyposis coli (APC) gene product. Many studies have shown that the b-catenin level is increased in cytoplasm and nucleus in various tumor types. The aim of this study was to evaluate b-catenin as a prognostic factor in SCC of the anal region. Methods: Immunohistochemical analysis of a tissue microarray with 52 squamous cell carcinoma specimens was performed to study the expression of b-catenin. Radiation alone or combined with chemotherapy (5-fluorouracil and mitomycin C) were given to all the patients except 4 of them who had local resections only. The Q-score method was used for evaluating membrane, cytoplasm and nucleus staining. The median follow-up was 4.8 years and the prognosis was analyzed using the Kaplan–Meier (KM) method and logrank test. The cut-off values were calculated using the receiver operating characteristics (ROC) curve. Results: Cytoplasmic overexpression of b-catenin was related to higher 5 years cancer specific survival (CSS) (79.2% vs. 39.3%, P = 0.005). The disease free survival (DFS) shows statistically significant correlation with high cytoplasmic expression and better survival (P = 0.009). Moreover, membranous expression of b-catenin was significantly correlated with higher survival rate (71.4% vs. 41.7%, P = 0.023) in CSS and (P = 0.024) in DFS. Clinicopathological analyses showed an association between the TN stadium and cytoplasmic-, but not membranous b-catenin. Nuclear expression of b-catenin was not detected. Conclusions: High cytoplasmic- and membranous b-catenin expression is associated with better prognosis in SCC of anal cancer patients. The b-catenin level in cytoplasm is normally bound to- and regulated by the APC complex. This regulation is mediated by b-catenin and E-cadherin binding at the membrane. The increased level of b-catenin might be a result of its abnormal regulation. However, further studies on other cytoplasmic and membranous candidates involved in the b-catenin pathway are needed to understand the mechanism where b-catenin is increased in cytoplasm and membrane of tumors. No conflict of interest. 502 AKT inhibition induces a prosurvival autophagy response that limits combination effects with gefitinib S.M. Bokobza1 , Y. Jiang1 , A.M. Devery1 , A.M. Weber1 , A.J. Ryan1 . 1 University of Oxford, Oncology, Oxford, United Kingdom Background: Although non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumours initially respond well to EGFR tyrosine kinase inhibitor (TKI) monotherapy, the benefits are usually limited due to the emergence of drug resistance. There is therefore significant unmet clinical need to improve efficacy of treatment for this group of NSCLC patients. Materials and Methods: NSCLC cells PC-9 (EGFR M+), HCC827 (EGFR M+), H1975 (EGFR T790M), and A549 (EGFR WT) were treated with MK2206 (1mM), AKT1/2 inhibitor (124018, Calbiochem, 1mM), or AKT2 inhibitor (124029, Calbiochem, 1mM), alone or in combination with gefitinib (0.1/1mM) and/or chloroquine (20mM) for 18 h, before Hoechst 33258 (25mM) was added, and cells analysed for apoptosis using the IN Cell analyzer 1000. The treated cells were also assessed for proliferation, PARP cleavage and LC3 levels by western blotting, as well as IF staining of LC3. For in vivo studies, Balb/C female nude mice (n = 36) were implanted with HCC827 cells (5×106 ). Once tumours reached 100 mm3 in volume, mice were treated with vehicle control (p.o., days 1−3), gefitinib (p.o. 25 mg/kg, days 1−3), MK2206 (p.o. 50 mg/kg, days 1−3), chloroquine (i.p. 60 mg/kg, days 1−3), gefitinib and chloroquine, MK2206 and chloroquine, gefitinib and MK2206, or gefitinib, MK2206, and chloroquine.
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Results: Our results show that treatment with selective AKT inhibitors (MK2206, AKT1/2 inhibitor, and AKT2 inhibitor) failed to induce apoptosis in NSCLC cells, but led to increased autophagy. When combined with EGFR TKI gefitinib, AKT inhibition synergistically decreased proliferation and increased apoptotic cell killing in EGFR M+ NSCLC cells. However, our data also suggest that the synergistic pro-apoptotic effects may be blunted due to a prosurvival autophagy response induced by AKT inhibition. Consequently, inhibiting autophagy with chloroquine significantly enhanced tumour cell death induced by gefitinib and AKT inhibitors in EGFR M+ cells in vitro, and produced greater tumour shrinkage in EGFR M+ xenografts in vivo. Conclusions: Together, our findings suggest that combining AKT and EGFR inhibition in EGFR M+ cells is beneficial, but may be limited by a prosurvival autophagy response. In support of this concept, adding chloroquine to EGFR and AKT inhibition produced greater tumour shrinkage in vivo, and we believe that this novel treatment has the potential to improve tumour responses in EGFR M+ NSCLC patients. No conflict of interest. 503 Novel FLT3 mutation shows dominant negative effects on the wild-type FLT3 receptor J. Bauer1,2 , N. Sandhofer ¨ 1,2,3 , W. Hiddemann1,2,3 , K. Spiekermann1,2,3 . 1 Clinical Cooperative Group Leukemia, Helmholtz Center Munich for Environmental Health, Munich, Germany, 2 Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University (LMU), Germany, 3 German Cancer Consortium (DKTK), Heidelberg, Germany, 4 German Cancer Research Center (DKFZ), Heidelberg, Germany Background: The FMS-like tyrosine kinase-3 (FLT3) gene encodes for a receptor tyrosine kinase playing an important role in hematopoiesis. In acute leukemias it is one of the most frequently mutated genes. In this study we functionally characterized a novel frameshift deletion mutation of FLT3 found in a relapsed patient with acute myeloid leukemia (AML). The frameshift leads to a premature stop codon resulting in a truncated form of the receptor lacking most of the intracellular domains. Material and Methods: FLT3 cDNA was expressed in the IL-3 dependent pro-B cell line Ba/F3 via a retroviral expression vector. The transduced cell lines were sorted by fluorescence-activated cell sorting (FACS). Stable expression of the receptor was confirmed on mRNA level by polymerase chain reaction (PCR) and on protein level by western blotting and cell surface expression of the receptor by flow cytometry. Cell proliferation assays were performed in presence or absence of IL-3 or FLT3-ligand. By western blotting receptor activation and its downstream signaling were analyzed. Ligand-binding of the receptor was shown via biotinylated and fluorescent FLT3-ligand-receptorcomplexes by flow cytometry. Results and Discussion: Stable expression of FLT3 Q569Vstop does not enable Ba/F3 cells to grow IL-3-independent. FLT3-ligand can still be bound by the mutated receptor but is not able to stimulate receptors’ signaling and growth of the cells. Furthermore coexpression of wild-type (WT) and mutant FLT3 receptor also abolishes the ability to stimulate the WT receptor with its ligand. This is confirmed by analyzing downstream signaling in the cells as MAPK is less phosphorylated in the FLT3-WT/Q569Vstop coexpressing cells than in FLT3-WT expressing cells alone. Conclusion: Most of the FLT3 mutations are activating mutations leading to a constitutive activation of the receptor and ligand-independent growth. In our study we characterized a novel FLT3 mutation found in an AML patient which has not been described before. The resulting truncated receptor is still integrated in the plasma membrane and binds its ligand but its ability to be fully activated is completely lost. Furthermore coexpressed with a FLT3-WT receptor, it even prevents stimulation and activation of the WT receptor, thus acting in a dominant negative manner. How the truncated form of the receptor contributes to progression of acute leukemia is of great interest and will be further investigated. No conflict of interest. 504 Functional proteomic analysis of leukaemogenic protein tyrosine kinase targets N. Che Azmi1 , A. Pierce1 , A. Williamson1 , A. Whetton1 . 1 University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom Background: The mutated thrombopoietin receptor MPL W515L found in thrombocytosis and myelofibrosis is constitutively activated leading to downstream signal transduction cascade activation including the Jak-Stat signaling pathway. Plainly oncogenes associated with myeloproliferative neoplasms (MPNs) have differential pathological effects. Using a proteomics approach we have investigated the differential effects of MPL W515L and other oncogenes associated with myeloproliferative neoplasms. These data have been employed to identify unique features of MPL W515L action that may account for its effects on the bone marrow microenvironment.
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Aims: To delineate the specific features of MPL W515L action with a view to identifying new therapeutic targets for MPN patients. Material and Methods: Cellular fractionation was undertaken with a kit from Active Motif (Rixensart, Belgium) with some modifications. Nuclear proteins of MPL W515L expressing cells and control were trypsin digested prior to labelling with isobaric tagging using 8 channel ITRAQ™ reagent (Applied Biosystems, MA) followed by phosphopeptide enrichment on TiO2 columns. Prior to reversed phase LC-MS/MS peptides were fractionated off line using a reversed phase chromatography column at a high pH using LC Packings Ultimate LC system. The data were analysed using Protein Pilot software 3 (ABSciex, UK). Western blotting was performed using standard protocols. The effect of inhibitor treatment on chemokinesis in MPL W515L expressing cells was measured via Boyden chamber assays. The levels of TGFb in the extracellular media were measured in cell culture supernatants using the ELISA assays. Results and Discussion: We have systematically analysed the proteomic effects of MPL W515L using a mass spectrometric approach. Within the proteins identified as changing due to MPL W515L expression, we saw an enrichment of proteins involved in motility. Further analysis of these data sets indicated a role for THOC5, a member of the THO complex involved in mRNA splicing/export pathway in the transformation process. Subsequent validation work elucidated a novel pathway of CXCL12/CD45 mediated Src activation of THOC5 Y225 which was linked to changes in cell motility. We have previously reported that TGFb is up-regulated by leukaemic oncogenes. Given its important in MPN myelofibrosis and motility, we investigated TGFb secretion in MPL W515L expressing cells. ELISA assessment of the secreted levels of TGFb showed a marked increase in MPL W515L expressing cells which was shown to contribute to enhanced chemokinesis. Addition of TGFb inhibitor to the MPL W515L cells led a significant reduction in chemokinesis. Furthermore western blot result showed down-regulation in the THOC5 Y225 phosphorylation in MPL W515L expressing cells treated with TGFb inhibitor. Thus MPL W515L oncogene induces a paracrine/autocrine set of effects via increased secretion of TGF beta leading to modulation of THOC5 phosphorylation (a regulator of mRNA usage) and motility in cell line studies. Conclusion: THOC5 is a downstream target of MPL W515L and its phosphorylation can affect motility. We have successfully delineated the novel signaling pathway of THOC5 and MPL W515L with TGFb and its effects on motility. No conflict of interest. 505 Reverse phase protein array profiling of lobular and triple negative breast cancers within the RATHER consortium L. De Koning1 , B. He1 , A. Barbet1 , F. Bard1 , C. Lecerf1 , C. Baldeyron1 , T. Dubois1 . 1 Institut Curie, Department of Translational Research, Paris, France Introduction: RATHER (Rational Therapy for Breast Cancer) is an international multi-site collaborative consortium that aims to use high resolution molecular profiling techniques to identify novel kinase targets for two subtypes of breast cancer, invasive lobular carcinomas (ILC) and triple negatives (TN), where no targeted therapies are currently available. Material and Method: DNA, RNA and protein were extracted from 137 ILC and 155 TN samples with a minimum of 5 years clinical follow-up. High resolution molecular profiling methods were used, including copy number analysis, gene expression profiling, targeted sequencing of the kinome, whole transcriptomic sequencing and reverse phase protein array (RPPA) analysis. Results and Discussion: Here, we will focus on the results obtained by RPPA analysis. First, we searched for proteins and signaling pathways that differentiate the TN and lobular carcinomas. Both expected and novel proteins/pathways were identified. Based on RPPA data, the lobular breast cancers can be subdivided into 4 groups characterized by distinct signaling pathway activation. These groups do not overlap with subgroups identified by transcriptome data and thus suggest that the proteomics data can provide additional information. In addition, we identify proteins that are associated with clinical outcome in a multivariate model taking into account known prognostic clinical variables such as tumor size, grade and treatment. These proteins thus represent potential biomarkers of interest in lobular carcinoma. Finally, we searched for proteins and pathways that characterize those tumors showing a high rate of mutations in the kinome, and those that are associated with CDH1 (E-cadherin) mutations, a marker of bad prognosis in lobular carcinomas. Conclusion: The RATHER project aims to identify kinase driver genes in both ILC and TN breast cancers to allow for the discovery of novel therapeutic targets. Integration of RPPA data with the other sources of molecular profiling data should allow for a subtype specific prognostic signature to be derived and the discovery of biomarkers for both diagnostic and prognostic purposes. No conflict of interest.
506 Relevance of BRAFG615E and BRAFG474R mutations in undifferentiated thyroid carcinomas R. Munoz ˜ Martinez1 , G. Fernandez ´ Ballester2 , A.M. Costa3 , T. Alvarez Gago4 , G. Garcia-Rostan1 . 1 Institute of Molecular Biology and Genetics (IBGM), Molecular Genetics of Disease, Vallodolid, Spain, 2 Institute of ´ Molecular and Cellular Biology (IBMC), Miguel Hernandez University, Elche − Alicante, Spain, 3 Institute of Molecular Pathology and Immunology of Porto University (IPATIMUP), Molecular Targets in Cancer, Porto, Portugal, 4 School of Medicine − Valladolid University, Department of Pathology, Valladolid, Spain The BRAFV600E mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency, different missense point mutations (K601E, G474R, G469R) and complex genetic alterations, such as rearrangements (AKAP9-BRAF) or frameshift mutations (V600EK601del, V599ins, V600D-FLAGT601–605ins, T599I-VKSRdel), have also been reported in PTC. In contrast with PTC, no other alteration different from the V600E substitution has been shown in anaplastic thyroid carcinomas (ATC). In this study, we sought to determine the functional relevance of particular aminoacid residues, within the BRAF kinase domain, that we found mutated in some ATC. Fifty-four ATC were investigated for the existence of mutations at the activation loop, the glicine-rich phosphate-binding loop and the AKT binding sites of BRAF by means of PCR-SSCP and direct sequence. The structuralconformational changes behind punctual, unreported BRAF mutations and their effect on BRAF catalytic activity, substrate binding and downstream MAPK signalling were evaluated. Besides the V600E mutation, two additional point mutations (G615E and G474R) were detected within the BRAF kinase domain. The prevalence of both mutations, G615E (11% = 6 ATC) and G474R (7.4% = 4 ATC), was much lower than that of V600E (20% = 11 ATC). Interestingly, structural biology studies showed that G615E and G474R induced profound conformational changes within the BRAF kinase domain that resulted in reduced catalytic activity, decreased substrate binding and impaired downstream signalling. The G474R mutant, by preventing the N lobe from folding into a well ordered beta sheet structure, caused a broken or distorted phosphorylation-loop (P-loop). Because the P-loop is pivotal for ATP coordination and phosphotransfer reactions, the kinase activity of the BRAF G474R mutant was severely compromised and BRAF was unable to phosphorylate the downstream substrate. The G615E mutant by disrupting the structural integrity of the activation segment in the C lobe could in addition inhibit substrate binding. Significantly, none of the ATC bearing the G615E or the G474R mutation expressed the phospho-p44/42 MAPK (Thr202/Tyr204), which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This observation not only supported the existence of impaired BRAF kinase activity in vivo but also suggested the abrogation of intracellular MEK/ERK signalling in those tumours. In summary, this study demonstrates that G615E and G474R behave similarly to D594V and the kinase-dead BRAFK483M mutant, knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. Clinical trials involving the inhibitor of threonine/serine kinases Sorafenib or kinase specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with G615E or G474R mutations. No conflict of interest. 507 The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin b8 in prostate cancer cells A. Herington1 , I. Mertens-Walker1 , B. Fernandini1 , A. Rockstroh1 , C. Nelson1 , S. Stephenson1 . 1 Institute of Health and Biomedical Innovation Qld Univ Technology, Australian Prostate Cancer Research Centre-Qld, Brisbane, Australia Background: EphB4 is a receptor tyrosine kinase that is over-expressed in 66% of prostate cancers and has been shown to play vital roles in cell migration and invasion in a variety of epithelial tumours. It is recognized as a potentially important therapeutic target. Little is known about the intrinsic pathways by which EphB4 promotes tumour progression. A key objective of this study was to define the molecular networks that the EphB4 receptor influences in prostate cancer. Material and Methods: We employed transient knock down of EphB4 in LNCaP cells followed by cDNA microarray analysis, coupled with bioinformatic approaches, to identify genes regulated by loss of EphB4. Validation experiments were carried out on selected target genes using quantitative realtime PCR and western immunoblotting to confirm their differential, EphB4mediated expression. Results: The microarray analysis revealed that 260 genes were upregulated and 300 were down-regulated when EphB4 was knocked down (by 70%) in LNCaP prostate cancer cells. Gene ontology analysis showed the process of cell adhesion as being most significantly influenced. Several integrins appeared to be deregulated, but Integrin b8 (ITGB8) was the top hit with a 29-
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Results: Our results show that treatment with selective AKT inhibitors (MK2206, AKT1/2 inhibitor, and AKT2 inhibitor) failed to induce apoptosis in NSCLC cells, but led to increased autophagy. When combined with EGFR TKI gefitinib, AKT inhibition synergistically decreased proliferation and increased apoptotic cell killing in EGFR M+ NSCLC cells. However, our data also suggest that the synergistic pro-apoptotic effects may be blunted due to a prosurvival autophagy response induced by AKT inhibition. Consequently, inhibiting autophagy with chloroquine significantly enhanced tumour cell death induced by gefitinib and AKT inhibitors in EGFR M+ cells in vitro, and produced greater tumour shrinkage in EGFR M+ xenografts in vivo. Conclusions: Together, our findings suggest that combining AKT and EGFR inhibition in EGFR M+ cells is beneficial, but may be limited by a prosurvival autophagy response. In support of this concept, adding chloroquine to EGFR and AKT inhibition produced greater tumour shrinkage in vivo, and we believe that this novel treatment has the potential to improve tumour responses in EGFR M+ NSCLC patients. No conflict of interest. 503 Novel FLT3 mutation shows dominant negative effects on the wild-type FLT3 receptor J. Bauer1,2 , N. Sandhofer ¨ 1,2,3 , W. Hiddemann1,2,3 , K. Spiekermann1,2,3 . 1 Clinical Cooperative Group Leukemia, Helmholtz Center Munich for Environmental Health, Munich, Germany, 2 Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University (LMU), Germany, 3 German Cancer Consortium (DKTK), Heidelberg, Germany, 4 German Cancer Research Center (DKFZ), Heidelberg, Germany Background: The FMS-like tyrosine kinase-3 (FLT3) gene encodes for a receptor tyrosine kinase playing an important role in hematopoiesis. In acute leukemias it is one of the most frequently mutated genes. In this study we functionally characterized a novel frameshift deletion mutation of FLT3 found in a relapsed patient with acute myeloid leukemia (AML). The frameshift leads to a premature stop codon resulting in a truncated form of the receptor lacking most of the intracellular domains. Material and Methods: FLT3 cDNA was expressed in the IL-3 dependent pro-B cell line Ba/F3 via a retroviral expression vector. The transduced cell lines were sorted by fluorescence-activated cell sorting (FACS). Stable expression of the receptor was confirmed on mRNA level by polymerase chain reaction (PCR) and on protein level by western blotting and cell surface expression of the receptor by flow cytometry. Cell proliferation assays were performed in presence or absence of IL-3 or FLT3-ligand. By western blotting receptor activation and its downstream signaling were analyzed. Ligand-binding of the receptor was shown via biotinylated and fluorescent FLT3-ligand-receptorcomplexes by flow cytometry. Results and Discussion: Stable expression of FLT3 Q569Vstop does not enable Ba/F3 cells to grow IL-3-independent. FLT3-ligand can still be bound by the mutated receptor but is not able to stimulate receptors’ signaling and growth of the cells. Furthermore coexpression of wild-type (WT) and mutant FLT3 receptor also abolishes the ability to stimulate the WT receptor with its ligand. This is confirmed by analyzing downstream signaling in the cells as MAPK is less phosphorylated in the FLT3-WT/Q569Vstop coexpressing cells than in FLT3-WT expressing cells alone. Conclusion: Most of the FLT3 mutations are activating mutations leading to a constitutive activation of the receptor and ligand-independent growth. In our study we characterized a novel FLT3 mutation found in an AML patient which has not been described before. The resulting truncated receptor is still integrated in the plasma membrane and binds its ligand but its ability to be fully activated is completely lost. Furthermore coexpressed with a FLT3-WT receptor, it even prevents stimulation and activation of the WT receptor, thus acting in a dominant negative manner. How the truncated form of the receptor contributes to progression of acute leukemia is of great interest and will be further investigated. No conflict of interest. 504 Functional proteomic analysis of leukaemogenic protein tyrosine kinase targets N. Che Azmi1 , A. Pierce1 , A. Williamson1 , A. Whetton1 . 1 University of Manchester, Wolfson Molecular Imaging Centre, Manchester, United Kingdom Background: The mutated thrombopoietin receptor MPL W515L found in thrombocytosis and myelofibrosis is constitutively activated leading to downstream signal transduction cascade activation including the Jak-Stat signaling pathway. Plainly oncogenes associated with myeloproliferative neoplasms (MPNs) have differential pathological effects. Using a proteomics approach we have investigated the differential effects of MPL W515L and other oncogenes associated with myeloproliferative neoplasms. These data have been employed to identify unique features of MPL W515L action that may account for its effects on the bone marrow microenvironment.
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Aims: To delineate the specific features of MPL W515L action with a view to identifying new therapeutic targets for MPN patients. Material and Methods: Cellular fractionation was undertaken with a kit from Active Motif (Rixensart, Belgium) with some modifications. Nuclear proteins of MPL W515L expressing cells and control were trypsin digested prior to labelling with isobaric tagging using 8 channel ITRAQ™ reagent (Applied Biosystems, MA) followed by phosphopeptide enrichment on TiO2 columns. Prior to reversed phase LC-MS/MS peptides were fractionated off line using a reversed phase chromatography column at a high pH using LC Packings Ultimate LC system. The data were analysed using Protein Pilot software 3 (ABSciex, UK). Western blotting was performed using standard protocols. The effect of inhibitor treatment on chemokinesis in MPL W515L expressing cells was measured via Boyden chamber assays. The levels of TGFb in the extracellular media were measured in cell culture supernatants using the ELISA assays. Results and Discussion: We have systematically analysed the proteomic effects of MPL W515L using a mass spectrometric approach. Within the proteins identified as changing due to MPL W515L expression, we saw an enrichment of proteins involved in motility. Further analysis of these data sets indicated a role for THOC5, a member of the THO complex involved in mRNA splicing/export pathway in the transformation process. Subsequent validation work elucidated a novel pathway of CXCL12/CD45 mediated Src activation of THOC5 Y225 which was linked to changes in cell motility. We have previously reported that TGFb is up-regulated by leukaemic oncogenes. Given its important in MPN myelofibrosis and motility, we investigated TGFb secretion in MPL W515L expressing cells. ELISA assessment of the secreted levels of TGFb showed a marked increase in MPL W515L expressing cells which was shown to contribute to enhanced chemokinesis. Addition of TGFb inhibitor to the MPL W515L cells led a significant reduction in chemokinesis. Furthermore western blot result showed down-regulation in the THOC5 Y225 phosphorylation in MPL W515L expressing cells treated with TGFb inhibitor. Thus MPL W515L oncogene induces a paracrine/autocrine set of effects via increased secretion of TGF beta leading to modulation of THOC5 phosphorylation (a regulator of mRNA usage) and motility in cell line studies. Conclusion: THOC5 is a downstream target of MPL W515L and its phosphorylation can affect motility. We have successfully delineated the novel signaling pathway of THOC5 and MPL W515L with TGFb and its effects on motility. No conflict of interest. 505 Reverse phase protein array profiling of lobular and triple negative breast cancers within the RATHER consortium L. De Koning1 , B. He1 , A. Barbet1 , F. Bard1 , C. Lecerf1 , C. Baldeyron1 , T. Dubois1 . 1 Institut Curie, Department of Translational Research, Paris, France Introduction: RATHER (Rational Therapy for Breast Cancer) is an international multi-site collaborative consortium that aims to use high resolution molecular profiling techniques to identify novel kinase targets for two subtypes of breast cancer, invasive lobular carcinomas (ILC) and triple negatives (TN), where no targeted therapies are currently available. Material and Method: DNA, RNA and protein were extracted from 137 ILC and 155 TN samples with a minimum of 5 years clinical follow-up. High resolution molecular profiling methods were used, including copy number analysis, gene expression profiling, targeted sequencing of the kinome, whole transcriptomic sequencing and reverse phase protein array (RPPA) analysis. Results and Discussion: Here, we will focus on the results obtained by RPPA analysis. First, we searched for proteins and signaling pathways that differentiate the TN and lobular carcinomas. Both expected and novel proteins/pathways were identified. Based on RPPA data, the lobular breast cancers can be subdivided into 4 groups characterized by distinct signaling pathway activation. These groups do not overlap with subgroups identified by transcriptome data and thus suggest that the proteomics data can provide additional information. In addition, we identify proteins that are associated with clinical outcome in a multivariate model taking into account known prognostic clinical variables such as tumor size, grade and treatment. These proteins thus represent potential biomarkers of interest in lobular carcinoma. Finally, we searched for proteins and pathways that characterize those tumors showing a high rate of mutations in the kinome, and those that are associated with CDH1 (E-cadherin) mutations, a marker of bad prognosis in lobular carcinomas. Conclusion: The RATHER project aims to identify kinase driver genes in both ILC and TN breast cancers to allow for the discovery of novel therapeutic targets. Integration of RPPA data with the other sources of molecular profiling data should allow for a subtype specific prognostic signature to be derived and the discovery of biomarkers for both diagnostic and prognostic purposes. No conflict of interest.
506 Relevance of BRAFG615E and BRAFG474R mutations in undifferentiated thyroid carcinomas R. Munoz ˜ Martinez1 , G. Fernandez ´ Ballester2 , A.M. Costa3 , T. Alvarez Gago4 , G. Garcia-Rostan1 . 1 Institute of Molecular Biology and Genetics (IBGM), Molecular Genetics of Disease, Vallodolid, Spain, 2 Institute of ´ Molecular and Cellular Biology (IBMC), Miguel Hernandez University, Elche − Alicante, Spain, 3 Institute of Molecular Pathology and Immunology of Porto University (IPATIMUP), Molecular Targets in Cancer, Porto, Portugal, 4 School of Medicine − Valladolid University, Department of Pathology, Valladolid, Spain The BRAFV600E mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency, different missense point mutations (K601E, G474R, G469R) and complex genetic alterations, such as rearrangements (AKAP9-BRAF) or frameshift mutations (V600EK601del, V599ins, V600D-FLAGT601–605ins, T599I-VKSRdel), have also been reported in PTC. In contrast with PTC, no other alteration different from the V600E substitution has been shown in anaplastic thyroid carcinomas (ATC). In this study, we sought to determine the functional relevance of particular aminoacid residues, within the BRAF kinase domain, that we found mutated in some ATC. Fifty-four ATC were investigated for the existence of mutations at the activation loop, the glicine-rich phosphate-binding loop and the AKT binding sites of BRAF by means of PCR-SSCP and direct sequence. The structuralconformational changes behind punctual, unreported BRAF mutations and their effect on BRAF catalytic activity, substrate binding and downstream MAPK signalling were evaluated. Besides the V600E mutation, two additional point mutations (G615E and G474R) were detected within the BRAF kinase domain. The prevalence of both mutations, G615E (11% = 6 ATC) and G474R (7.4% = 4 ATC), was much lower than that of V600E (20% = 11 ATC). Interestingly, structural biology studies showed that G615E and G474R induced profound conformational changes within the BRAF kinase domain that resulted in reduced catalytic activity, decreased substrate binding and impaired downstream signalling. The G474R mutant, by preventing the N lobe from folding into a well ordered beta sheet structure, caused a broken or distorted phosphorylation-loop (P-loop). Because the P-loop is pivotal for ATP coordination and phosphotransfer reactions, the kinase activity of the BRAF G474R mutant was severely compromised and BRAF was unable to phosphorylate the downstream substrate. The G615E mutant by disrupting the structural integrity of the activation segment in the C lobe could in addition inhibit substrate binding. Significantly, none of the ATC bearing the G615E or the G474R mutation expressed the phospho-p44/42 MAPK (Thr202/Tyr204), which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This observation not only supported the existence of impaired BRAF kinase activity in vivo but also suggested the abrogation of intracellular MEK/ERK signalling in those tumours. In summary, this study demonstrates that G615E and G474R behave similarly to D594V and the kinase-dead BRAFK483M mutant, knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. Clinical trials involving the inhibitor of threonine/serine kinases Sorafenib or kinase specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with G615E or G474R mutations. No conflict of interest. 507 The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin b8 in prostate cancer cells A. Herington1 , I. Mertens-Walker1 , B. Fernandini1 , A. Rockstroh1 , C. Nelson1 , S. Stephenson1 . 1 Institute of Health and Biomedical Innovation Qld Univ Technology, Australian Prostate Cancer Research Centre-Qld, Brisbane, Australia Background: EphB4 is a receptor tyrosine kinase that is over-expressed in 66% of prostate cancers and has been shown to play vital roles in cell migration and invasion in a variety of epithelial tumours. It is recognized as a potentially important therapeutic target. Little is known about the intrinsic pathways by which EphB4 promotes tumour progression. A key objective of this study was to define the molecular networks that the EphB4 receptor influences in prostate cancer. Material and Methods: We employed transient knock down of EphB4 in LNCaP cells followed by cDNA microarray analysis, coupled with bioinformatic approaches, to identify genes regulated by loss of EphB4. Validation experiments were carried out on selected target genes using quantitative realtime PCR and western immunoblotting to confirm their differential, EphB4mediated expression. Results: The microarray analysis revealed that 260 genes were upregulated and 300 were down-regulated when EphB4 was knocked down (by 70%) in LNCaP prostate cancer cells. Gene ontology analysis showed the process of cell adhesion as being most significantly influenced. Several integrins appeared to be deregulated, but Integrin b8 (ITGB8) was the top hit with a 29-