- Email: [email protected]
506: Relevance of BRAFG615E and BRAFG474R mutations in undifferentiated thyroid carcinomas
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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-
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-