- Email: [email protected]
363: The ING1 tumour suppressor induces senescence via altering endocytosis
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
surrounding tissue, indicating that the polarity protein Par3 counteracts disease progression through control of proper tissue integrity. Conclusion: We identified crucial roles of conserved regulators of polarity in keratinocyte proliferation and apoptosis, and demonstrated that impaired Par3 function affects skin tumor onset and progression. We are further investigating how the cortical Par3 complex links cell polarity with key oncogenic signaling pathways driving non-melanoma skin cancers. These studies will help to provide valuable insight into the molecular coupling of cyto-architecture and tissue homeostasis by polarity proteins − findings that may prove important to prevent formation and progression of cancer. No conflict of interest. 360 Reduced promoter methylation and increased expression of CSPG4 negatively influences survival of HNSCC patients R.W. Warta1 , J.C. Chaisaingmongkol2 , A.M. Mock3 , O.P. Popanda2 , E.H. Herpel4 , C.P. Plass2 , P.S. Schmezer2 , V.E. Eckstein5 , G.D. Dyckhoff1 , C.H.M. Herold-Mende3 . 1 University Hospital Heidelberg, Department of Otorhinolaryngology Head and Neck Surgery, Heidelberg, Germany, 2 German Cancer Research Center, Division of Epigenomics and Cancer Risk Factors, Heidelberg, Germany, 3 University Hospital Heidelberg, Division of Experimental Neurosurgery Department of Neurosurgery, Heidelberg, Germany, 4 National Center for Tumor Diseases, NCT Tissue Bank, Heidelberg, Germany, 5 University Hospital Heidelberg, Department of Medicine V, Heidelberg, Germany Background: HNSCC biology is strongly influenced by various genetic alterations that appear in early tumor stages. Moreover there is now increasing evidence that epigenetic changes like DNA methylation also contribute significantly to malignant transformation. The expression level of markers of undifferentiated cells might be accurate prognostic indicators in cancer. Interestingly, there are hints that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on normal neural stem cells and cancer stem cells of gliomas and its expression is associated with a poor prognosis and resistance to treatment. Therefore we hypothesize that CSPG4 is a good candidate HNSCC marker, however a systematic analysis of the role of CSPG4 in HNSCC is still missing. Material and Methods: We performed an in-depth analysis of CSPG4 expression in a panel of HNSCC tumors and normal controls. Therefore we used immunohistochemical staining, qPCR and in silico expression analysis of public available expression microarray datasets. The DNA-methylation status of a CpG-Island in CSPG4 promoter in HNSCC tumors was analyzed by the highly innovative MassARRAY technique and validated by 5-AZA treatment of two HNSCC-cell lines. Finally the CSPG4 expression data and the methylation status of the CSPG4 promoter were correlated to the survival prognosis of the HNSCC patients. Results: The expression analysis revealed a dramatic CSPG4 mRNA overexpression in HNSCC tumors and an increased CSPG4 protein expression in a subgroup of HNSCCs. Next we identified hypomethylation of the CSPG4 promoter in HNSCC which correlated with high CSPG4 mRNA and protein expression. This was confirmed by 5-AZA treatment of two HNSCC celllines. Finally we found that high CSPG4 expression and low promoter methylation are significantly associated with an adverse progression-free and overall survival. Conclusion: In this first in-depth study of CSPG4 in HNSCC we revealed, that overall as well as progression-free survival of HNSCC patients is linked to tumor CSPG4 promoter methylation and protein expression. We found that CSPG4 expression is often dramatically increased in HNSCC tumors, a result consistent with our detection of generally lower CSPG4 promoter methylation. These data suggest that in HNSCC tumor cells, demethylation at the CSPG4 promoter contributes to increased CSPG4 expression. In support of this possibility, we found that treating HNSCC cells harboring a highly methylated CSPG4 promoter with demethylation agents caused CSPG4 reexpression. In conclusion our findings support our initial hypothesis that CSPG4 is an important player in HNSCC biology. No conflict of interest. 361 The putative tumor suppressor gene Dickkopf-3 (DKK3) and its role in the carcinogenesis of breast cancer 1 E. Lorsy1 , J. Veeck1 , R. Knuchel ¨ , E. Dahl1 . 1 University Hospital RWTH Aachen, Pathology, Aachen, Germany
Introduction: The expression of the putative WNT signaling inhibitor Dickkopf-3 (DKK3) is downregulated in several tumor entities. Previously, we have shown that downregulation of DKK3 in human breast cancer is due to DKK3 promoter hypermethylation and that this molecular lesion is associated with unfavorable patient prognosis. In our current study, we are analyzing the biological function of DKK3 in human breast cancer cell lines. Materials and Methods: MDA-MB-231 and MDA-MB-436 basal-type as well as MCF7 and MDA-MB-453 luminal-type breast cancer cell lines, all with no endogenous DKK3 expression, were stably transfected using a fulllength DKK3
cDNA containing vector or empty vector. In vitro cell culture assays determined the impact on proliferation, colony formation and substrate adhesion. Results: Real-time PCR and Western blot analysis confirmed abundant reexpression of DKK3 mRNA and protein in DKK3-transfected MDA-MB-231, MDA-MB-436, MDA-MB-453 and MCF7 cells. MDA-MB-436 cells switched from a mesenchymal to a more epithelial morphology after transfection with DKK3 accompanied with increased expression of epithilial markers (e.g. Claudin 1 and ZO-1). DKK3 clones of both the luminal and the basal-type exhibited reduced growth capabilities in comparison with mock clones (p = 0.002 for MDA-MB-436 and p = 0.01 for MCF7). DKK3-clones of the basal-type cell line MDA-MB-436 attached significantly faster to BD Matrigel™ (Basement Membrane Matrix) than mock clones (p < 0.0001), while no differences were observed in luminal-type MCF7 cells (p > 0.05). This discrepancy will be further addressed with additional cell lines, as functional analyses with other cell lines and assays (e.g. apoptosis) are still ongoing. Conclusions: DKK3 may represent a novel tumor suppressor gene in normal breast tissue since DKK3 is capable of suppressing cell proliferation of human breast cancer cells in vitro and DKK3 promoter methylation in human tumors is associated with poor survival. Ongoing studies are analyzing the influence of DKK3 on apoptosis and invasion in vitro. In addition, in vivo experiments using dkk3-knockout mice will be performed. No conflict of interest. 362 BMP4 suppresses breast cancer metastasis through down-regulation of G-CSF R.L. Anderson1 , Y. Cao1 , A. Swierczak1 , B.L. Eckhardt2 , J.A. Hamilton3 . 1 Peter MacCallum Cancer Centre, Research Division, East Melbourne Victoria, Australia, 2 MD Anderson Cancer Center, Department of Breast Medical Oncology, Houston Texas, USA, 3 The University of Melbourne, Department of Medicine, Melbourne Victoria, Australia Background: Most deaths from breast cancer are due to the onset of metastatic disease, for which there are few curative therapies. A better understanding of molecular mechanisms driving metastasis offers the possibility of improved therapies. In our screens for metastasis regulators, we discovered that bone morphogenetic protein 4 (BMP4) is a powerful metastasis suppressor. BMP4 belongs to the transforming growth factor (TGF-b) superfamily and is an important cytokine regulating many biological functions in a context dependent manner. Materials and Methods: We have used preclinical models of spontaneous breast cancer metastasis to explore the mechanisms by which BMP4 regulates metastasis. We have also investigated the prognostic significance of BMP4 in tissue arrays of cancers from breast cancer patients. Results: High levels of BMP4 correlated with improved outcome in breast cancer patients. In our preclinical models, BMP4 did not alter primary tumour growth but profoundly reduced development of metastases. In therapy mode, recombinant BMP4 (rBMP4) prolonged survival of mice bearing highly metastatic mammary tumours. In seeking the mechanism by which BMP4 suppresses metastasis, we found that either expression of BMP4 in mammary tumour cells in vitro or treatment with rBMP4 led to down-regulation of G-CSF secretion. An analysis of plasma from mice bearing metastatic mammary tumours revealed high levels of G-CSF that were reduced in mice whose tumours expressed BMP4. In parallel with reduced G-CSF, we found a lower proportion of myeloid derived suppressor cells (MDSC) in peripheral blood. Direct injection of G-CSF into mice stimulated the mobilisation of MDSC and enhanced the metastatic capacity of mammary tumours. The MDSC induced by treatment of mice with G-CSF were able to suppress CD4+ /CD8+ T cell proliferation, demonstrating their immunosuppressive activity. In contrast, the MDSC present in mice bearing BMP4 expressing tumours had reduced immunosuppressive activity. We have demonstrated that BMP4 regulates G-CSF expression through inhibition of NFkB signaling. A direct impact of G-CSF signaling on metastasis was demonstrated by showing that treatment of tumour-bearing mice with a neutralising antibody against G-CSF receptor (G-CSFR) led to a reduction in MDSC and a reduction in spontaneous metastasis of mammary tumours to lung and bone. Conclusions: These observations open a path for a new therapy for advanced breast cancer based on activation of BMP4 signaling and/or blockade of GCSFR signaling. No conflict of interest. 363 The ING1 tumour suppressor induces senescence via altering endocytosis K. Riabowol1 , U.K. Rajarajacholan1 , S. Thaalippilly1 . 1 University of Calgary, Biochemistry & Molecular Biology and Oncology, Calgary Alberta, Canada Background: The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Alternative
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 splicing products of the ING1 gene affect apoptosis and cell senescence, but the mechanisms are currently unclear. Materials and Methods: Primary diploid fibroblasts were examined for their propensity to express different isoforms of ING1, and their response to increased and decreased levels of ING1 was examined. Results: Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated b-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1ainduced senescence signaling. Conclusions: These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype. Since ING1a levels increase naturally near the end of cell replicative activity, the alternative splicing of ING1 might represent a regulatory event that subsequently prevents senescing cells from responding normally to mitogenic factors, helping enforce the senescence phenotype believed to block the progression of cancer. No conflict of interest. 364 VHL-dependent changes in global kinome expression in renal cell carcinoma K. Rantanen1,2 , P. Kouvonen1 , G.L. Corthals1 , P.M. Jaakkola1,2,3 . 1 Turku ˚ Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland, 2 Institute of Biomedicine, Medical School, University of Turku, 3 Finland, Department of Oncology and Radiation Therapy, Turku University Hospital, Turku, Finland Background: Renal cell carcinoma (RCC) is the most common form of kidney cancer. Despite the advances in treating RCC, especially at metastatic stage the prognosis is still quite poor. In RCC, the inactivation of tumor suppressor gene vhl is the most frequent abnormality leading to the stabilization of HIF-1a. The constant activation of HIF-1a is strongly oncogenic and is essential for tumor growth. Therapeutically targeting an inhibiting kinase pathways like mTOR and VEGF by multikinase inhibitors are used for metastatic RCC. However, the biological phenomena behind RCC development is not fully understood. In this study we are aiming to delineate the effect of VHL mutation in global kinase expression in RCC. We are looking into the kinome variability in within VHL-defective RCC as well as between VHL-defective and VHL-restored contexts. Materials and Methods: We are utilizing two RCC cell linesnaturally occuring RCC4 with VHL mutation (RCC4 VHL−/− ) and RCC with restored VHL (RCCVHL+/+ ) in order to study the kinome expression regulation in normoxia and in hypoxia. Targeted mass spectrometry (Selected Reaction Monitoring, SRM) is used to monitor relative kinase abundances related to mutational status of VHL. SRM transitions for selected 77 kinases have been confirmed in a previous study on RCC cell lines and RCC patient tissue. Results and Discussion: Our data suggests a VHL-dependent differential expression of several kinases in renal cell carcinoma. Changes in the expression of kinases such as ROCK, EGFR and MAPK6 of others number were observed between RCCVHL−/− and RCCVHL+/+ . Conclusions: In this study we are characterising the effect of VHL inactivation on the expression of kinases. For some of these kinases the regulation is VHLdependent and for others hypoxia VHL-independent regulating factor. No conflict of interest. 365 The interplay between the redox environment, MTH1 and cancer L. Braeutigam1 , R. Fiskesund1 , U. Warpman-Berglund1 , T. Helleday1 . 1 Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Sweden Introduction: Redox regulation, the signal transduction through oxidation and reduction processes, is highly important for almost every aspect of cellular function. During the recent years it has become increasingly recognized that cancer cells have a distinct, deregulated redox profile compared to non-tumorigenic cells. Targeting those reactive-oxygen species mediated mechanisms specific for cancer cells might open new avenues for future
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therapies. We have exemplified the potential of this reasoning by developing inhibitors against MTH1 which detoxifies oxidized free nucleotides. Material and Methods: We study the relation between the redox environment, MTH1 and cancer using cell culture systems, mice and the zebrafish model organism. Amongst others, we challenge cells with different agents that change the cellular redox profile and use varying oxygen concentration to study its impact on drug sensitivity as well as tumorigenic potential. Results and Discussion: Due to the specific redox profile of cancer cells, their nucleotide pool is highly oxidized. This makes them dependent on MTH1, a Nudix-family protein which sanitizes oxidized nucleotides. We have recently shown that MTH1 is a new promising target for cancer therapy. Here, we present data that shines light on the intricate relation between MTH1, oxygen concentration and the redox environment of the cancer cell. Moreover, we present evidence that MTH1 is connected to distinct oxygen sensing and redox signaling pathways and how these connections might be used for future cancer therapy. Conclusion: Cancer cells are characterized by a redox environment that is clearly distinct from that of non-tumorigenic cells. We are exploiting the cancer-specific redox environment as drug target and are confident that this knowledge will lead to new therapeutical avenues as exemplified by our strategy targeting MTH1. No conflict of interest. 366 The role of tumour derived extracellular matrices on macrophage polarization M.L. Pinto1,2 , E. Rios3,4 , A. Silva1 , A.T. Pinto1,5 , A.P. Cardoso1,5 , ´ 1,5 , F. Carneiro3,4,6 , M.B. Barbosa1,2 , D. Nascimento1 , P. Pinto do O M.J. Oliveira1,3 . 1 INEB, Institute of Biomedical Engineering, Oporto, Portugal, 2 ICBAS-Abel Salazar Institute for Biomedical Sciences, Porto, Portugal, 3 Department of Pathology and Oncology from Faculty of Medicine, ˜ Joao ˜ Hospital Porto, Portugal, 4 Department of Anatomic Pathology, Sao Center, Porto, Portugal, 5 FEUP-Faculty of Engineering, Porto, Portugal, 6 IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, Portugal Introduction: Tumours are highly complex microecosystems composed of cancer cells, extracellular matrix (ECM) components and other cell types. The molecular crosstalks established between cancer cells and the surrounding environment are crucial for tumour progression. Macrophages have been described as key elements in this process, preventing the spreading of cancer cells − M1 macrophages − or supporting tumour progression − M2 macrophages. We are particularly interested to elucidate how the ECM and tumour cells contribute to macrophage polarization. Therefore, we are creating a 3D-organotypic culture mode, by decellularizing human colorectal cancer (CRC) tissue fragments and by repopulating them with monocytes and or/tumour cells, mimicking more closely the natural tumour microecosystem. Materials and Methods: We optimized the decellularization protocol and accessed its efficiency as well as tissue morphology and architecture by light microscopy and Scanning Electron Microscopy (SEM). The effect of tissue decellularization on DNA and Glycosaminoglycans (GAGs) contents were evaluated. These matrices were then repopulated with freshly isolated monocytes and allowed to differentiate for 7 days. Additionally, we characterized through immunohistochemistry (IHC) analysis, CD68, CD163, HLA-DR and calprotectin expression profile of macrophage populations in CRC cases from Sao ˜ Joao ˜ Hospital Center. Results and Discussion: Deoxyribonucleic acid (DNA) quantification and 4 ,6-diamidino-2-phenylindole (DAPI) staining confirmed the efficiency of the decellularization method. SEM analysis allowed the visualization of the ECM fiber meshwork while staining with Hematoxylin-Eosin (HE) and Masson’s Trichrome revealed that decellularized fragments retain the histological features of the tissues. Decellularization reduced significantly the GAGS content in normal and tumours but other ECM components, such as laminin or fibronectin, are retained. Preliminary results clearly evidenced that monocytes are able to colonize decellularized matrices and to differentiate into macrophages within the fiber network. Conclusion: The decellularization protocol is effective in decellularizing normal and tumour fragments while retaining, at least partially, tissue architecture and composition. Preliminary results on matrices repopulations suggest that this could be a helpful model to study tumour complexity. At completion of this project we expect to have elucidated the role of tumour cells and of ECM components, derived from the tumour microecosystem, on macrophage differentiation and polarization, contributing to the design of novel therapeutic strategies targeting macrophages. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
surrounding tissue, indicating that the polarity protein Par3 counteracts disease progression through control of proper tissue integrity. Conclusion: We identified crucial roles of conserved regulators of polarity in keratinocyte proliferation and apoptosis, and demonstrated that impaired Par3 function affects skin tumor onset and progression. We are further investigating how the cortical Par3 complex links cell polarity with key oncogenic signaling pathways driving non-melanoma skin cancers. These studies will help to provide valuable insight into the molecular coupling of cyto-architecture and tissue homeostasis by polarity proteins − findings that may prove important to prevent formation and progression of cancer. No conflict of interest. 360 Reduced promoter methylation and increased expression of CSPG4 negatively influences survival of HNSCC patients R.W. Warta1 , J.C. Chaisaingmongkol2 , A.M. Mock3 , O.P. Popanda2 , E.H. Herpel4 , C.P. Plass2 , P.S. Schmezer2 , V.E. Eckstein5 , G.D. Dyckhoff1 , C.H.M. Herold-Mende3 . 1 University Hospital Heidelberg, Department of Otorhinolaryngology Head and Neck Surgery, Heidelberg, Germany, 2 German Cancer Research Center, Division of Epigenomics and Cancer Risk Factors, Heidelberg, Germany, 3 University Hospital Heidelberg, Division of Experimental Neurosurgery Department of Neurosurgery, Heidelberg, Germany, 4 National Center for Tumor Diseases, NCT Tissue Bank, Heidelberg, Germany, 5 University Hospital Heidelberg, Department of Medicine V, Heidelberg, Germany Background: HNSCC biology is strongly influenced by various genetic alterations that appear in early tumor stages. Moreover there is now increasing evidence that epigenetic changes like DNA methylation also contribute significantly to malignant transformation. The expression level of markers of undifferentiated cells might be accurate prognostic indicators in cancer. Interestingly, there are hints that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on normal neural stem cells and cancer stem cells of gliomas and its expression is associated with a poor prognosis and resistance to treatment. Therefore we hypothesize that CSPG4 is a good candidate HNSCC marker, however a systematic analysis of the role of CSPG4 in HNSCC is still missing. Material and Methods: We performed an in-depth analysis of CSPG4 expression in a panel of HNSCC tumors and normal controls. Therefore we used immunohistochemical staining, qPCR and in silico expression analysis of public available expression microarray datasets. The DNA-methylation status of a CpG-Island in CSPG4 promoter in HNSCC tumors was analyzed by the highly innovative MassARRAY technique and validated by 5-AZA treatment of two HNSCC-cell lines. Finally the CSPG4 expression data and the methylation status of the CSPG4 promoter were correlated to the survival prognosis of the HNSCC patients. Results: The expression analysis revealed a dramatic CSPG4 mRNA overexpression in HNSCC tumors and an increased CSPG4 protein expression in a subgroup of HNSCCs. Next we identified hypomethylation of the CSPG4 promoter in HNSCC which correlated with high CSPG4 mRNA and protein expression. This was confirmed by 5-AZA treatment of two HNSCC celllines. Finally we found that high CSPG4 expression and low promoter methylation are significantly associated with an adverse progression-free and overall survival. Conclusion: In this first in-depth study of CSPG4 in HNSCC we revealed, that overall as well as progression-free survival of HNSCC patients is linked to tumor CSPG4 promoter methylation and protein expression. We found that CSPG4 expression is often dramatically increased in HNSCC tumors, a result consistent with our detection of generally lower CSPG4 promoter methylation. These data suggest that in HNSCC tumor cells, demethylation at the CSPG4 promoter contributes to increased CSPG4 expression. In support of this possibility, we found that treating HNSCC cells harboring a highly methylated CSPG4 promoter with demethylation agents caused CSPG4 reexpression. In conclusion our findings support our initial hypothesis that CSPG4 is an important player in HNSCC biology. No conflict of interest. 361 The putative tumor suppressor gene Dickkopf-3 (DKK3) and its role in the carcinogenesis of breast cancer 1 E. Lorsy1 , J. Veeck1 , R. Knuchel ¨ , E. Dahl1 . 1 University Hospital RWTH Aachen, Pathology, Aachen, Germany
Introduction: The expression of the putative WNT signaling inhibitor Dickkopf-3 (DKK3) is downregulated in several tumor entities. Previously, we have shown that downregulation of DKK3 in human breast cancer is due to DKK3 promoter hypermethylation and that this molecular lesion is associated with unfavorable patient prognosis. In our current study, we are analyzing the biological function of DKK3 in human breast cancer cell lines. Materials and Methods: MDA-MB-231 and MDA-MB-436 basal-type as well as MCF7 and MDA-MB-453 luminal-type breast cancer cell lines, all with no endogenous DKK3 expression, were stably transfected using a fulllength DKK3
cDNA containing vector or empty vector. In vitro cell culture assays determined the impact on proliferation, colony formation and substrate adhesion. Results: Real-time PCR and Western blot analysis confirmed abundant reexpression of DKK3 mRNA and protein in DKK3-transfected MDA-MB-231, MDA-MB-436, MDA-MB-453 and MCF7 cells. MDA-MB-436 cells switched from a mesenchymal to a more epithelial morphology after transfection with DKK3 accompanied with increased expression of epithilial markers (e.g. Claudin 1 and ZO-1). DKK3 clones of both the luminal and the basal-type exhibited reduced growth capabilities in comparison with mock clones (p = 0.002 for MDA-MB-436 and p = 0.01 for MCF7). DKK3-clones of the basal-type cell line MDA-MB-436 attached significantly faster to BD Matrigel™ (Basement Membrane Matrix) than mock clones (p < 0.0001), while no differences were observed in luminal-type MCF7 cells (p > 0.05). This discrepancy will be further addressed with additional cell lines, as functional analyses with other cell lines and assays (e.g. apoptosis) are still ongoing. Conclusions: DKK3 may represent a novel tumor suppressor gene in normal breast tissue since DKK3 is capable of suppressing cell proliferation of human breast cancer cells in vitro and DKK3 promoter methylation in human tumors is associated with poor survival. Ongoing studies are analyzing the influence of DKK3 on apoptosis and invasion in vitro. In addition, in vivo experiments using dkk3-knockout mice will be performed. No conflict of interest. 362 BMP4 suppresses breast cancer metastasis through down-regulation of G-CSF R.L. Anderson1 , Y. Cao1 , A. Swierczak1 , B.L. Eckhardt2 , J.A. Hamilton3 . 1 Peter MacCallum Cancer Centre, Research Division, East Melbourne Victoria, Australia, 2 MD Anderson Cancer Center, Department of Breast Medical Oncology, Houston Texas, USA, 3 The University of Melbourne, Department of Medicine, Melbourne Victoria, Australia Background: Most deaths from breast cancer are due to the onset of metastatic disease, for which there are few curative therapies. A better understanding of molecular mechanisms driving metastasis offers the possibility of improved therapies. In our screens for metastasis regulators, we discovered that bone morphogenetic protein 4 (BMP4) is a powerful metastasis suppressor. BMP4 belongs to the transforming growth factor (TGF-b) superfamily and is an important cytokine regulating many biological functions in a context dependent manner. Materials and Methods: We have used preclinical models of spontaneous breast cancer metastasis to explore the mechanisms by which BMP4 regulates metastasis. We have also investigated the prognostic significance of BMP4 in tissue arrays of cancers from breast cancer patients. Results: High levels of BMP4 correlated with improved outcome in breast cancer patients. In our preclinical models, BMP4 did not alter primary tumour growth but profoundly reduced development of metastases. In therapy mode, recombinant BMP4 (rBMP4) prolonged survival of mice bearing highly metastatic mammary tumours. In seeking the mechanism by which BMP4 suppresses metastasis, we found that either expression of BMP4 in mammary tumour cells in vitro or treatment with rBMP4 led to down-regulation of G-CSF secretion. An analysis of plasma from mice bearing metastatic mammary tumours revealed high levels of G-CSF that were reduced in mice whose tumours expressed BMP4. In parallel with reduced G-CSF, we found a lower proportion of myeloid derived suppressor cells (MDSC) in peripheral blood. Direct injection of G-CSF into mice stimulated the mobilisation of MDSC and enhanced the metastatic capacity of mammary tumours. The MDSC induced by treatment of mice with G-CSF were able to suppress CD4+ /CD8+ T cell proliferation, demonstrating their immunosuppressive activity. In contrast, the MDSC present in mice bearing BMP4 expressing tumours had reduced immunosuppressive activity. We have demonstrated that BMP4 regulates G-CSF expression through inhibition of NFkB signaling. A direct impact of G-CSF signaling on metastasis was demonstrated by showing that treatment of tumour-bearing mice with a neutralising antibody against G-CSF receptor (G-CSFR) led to a reduction in MDSC and a reduction in spontaneous metastasis of mammary tumours to lung and bone. Conclusions: These observations open a path for a new therapy for advanced breast cancer based on activation of BMP4 signaling and/or blockade of GCSFR signaling. No conflict of interest. 363 The ING1 tumour suppressor induces senescence via altering endocytosis K. Riabowol1 , U.K. Rajarajacholan1 , S. Thaalippilly1 . 1 University of Calgary, Biochemistry & Molecular Biology and Oncology, Calgary Alberta, Canada Background: The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Alternative
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 splicing products of the ING1 gene affect apoptosis and cell senescence, but the mechanisms are currently unclear. Materials and Methods: Primary diploid fibroblasts were examined for their propensity to express different isoforms of ING1, and their response to increased and decreased levels of ING1 was examined. Results: Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated b-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1ainduced senescence signaling. Conclusions: These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype. Since ING1a levels increase naturally near the end of cell replicative activity, the alternative splicing of ING1 might represent a regulatory event that subsequently prevents senescing cells from responding normally to mitogenic factors, helping enforce the senescence phenotype believed to block the progression of cancer. No conflict of interest. 364 VHL-dependent changes in global kinome expression in renal cell carcinoma K. Rantanen1,2 , P. Kouvonen1 , G.L. Corthals1 , P.M. Jaakkola1,2,3 . 1 Turku ˚ Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland, 2 Institute of Biomedicine, Medical School, University of Turku, 3 Finland, Department of Oncology and Radiation Therapy, Turku University Hospital, Turku, Finland Background: Renal cell carcinoma (RCC) is the most common form of kidney cancer. Despite the advances in treating RCC, especially at metastatic stage the prognosis is still quite poor. In RCC, the inactivation of tumor suppressor gene vhl is the most frequent abnormality leading to the stabilization of HIF-1a. The constant activation of HIF-1a is strongly oncogenic and is essential for tumor growth. Therapeutically targeting an inhibiting kinase pathways like mTOR and VEGF by multikinase inhibitors are used for metastatic RCC. However, the biological phenomena behind RCC development is not fully understood. In this study we are aiming to delineate the effect of VHL mutation in global kinase expression in RCC. We are looking into the kinome variability in within VHL-defective RCC as well as between VHL-defective and VHL-restored contexts. Materials and Methods: We are utilizing two RCC cell linesnaturally occuring RCC4 with VHL mutation (RCC4 VHL−/− ) and RCC with restored VHL (RCCVHL+/+ ) in order to study the kinome expression regulation in normoxia and in hypoxia. Targeted mass spectrometry (Selected Reaction Monitoring, SRM) is used to monitor relative kinase abundances related to mutational status of VHL. SRM transitions for selected 77 kinases have been confirmed in a previous study on RCC cell lines and RCC patient tissue. Results and Discussion: Our data suggests a VHL-dependent differential expression of several kinases in renal cell carcinoma. Changes in the expression of kinases such as ROCK, EGFR and MAPK6 of others number were observed between RCCVHL−/− and RCCVHL+/+ . Conclusions: In this study we are characterising the effect of VHL inactivation on the expression of kinases. For some of these kinases the regulation is VHLdependent and for others hypoxia VHL-independent regulating factor. No conflict of interest. 365 The interplay between the redox environment, MTH1 and cancer L. Braeutigam1 , R. Fiskesund1 , U. Warpman-Berglund1 , T. Helleday1 . 1 Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Sweden Introduction: Redox regulation, the signal transduction through oxidation and reduction processes, is highly important for almost every aspect of cellular function. During the recent years it has become increasingly recognized that cancer cells have a distinct, deregulated redox profile compared to non-tumorigenic cells. Targeting those reactive-oxygen species mediated mechanisms specific for cancer cells might open new avenues for future
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therapies. We have exemplified the potential of this reasoning by developing inhibitors against MTH1 which detoxifies oxidized free nucleotides. Material and Methods: We study the relation between the redox environment, MTH1 and cancer using cell culture systems, mice and the zebrafish model organism. Amongst others, we challenge cells with different agents that change the cellular redox profile and use varying oxygen concentration to study its impact on drug sensitivity as well as tumorigenic potential. Results and Discussion: Due to the specific redox profile of cancer cells, their nucleotide pool is highly oxidized. This makes them dependent on MTH1, a Nudix-family protein which sanitizes oxidized nucleotides. We have recently shown that MTH1 is a new promising target for cancer therapy. Here, we present data that shines light on the intricate relation between MTH1, oxygen concentration and the redox environment of the cancer cell. Moreover, we present evidence that MTH1 is connected to distinct oxygen sensing and redox signaling pathways and how these connections might be used for future cancer therapy. Conclusion: Cancer cells are characterized by a redox environment that is clearly distinct from that of non-tumorigenic cells. We are exploiting the cancer-specific redox environment as drug target and are confident that this knowledge will lead to new therapeutical avenues as exemplified by our strategy targeting MTH1. No conflict of interest. 366 The role of tumour derived extracellular matrices on macrophage polarization M.L. Pinto1,2 , E. Rios3,4 , A. Silva1 , A.T. Pinto1,5 , A.P. Cardoso1,5 , ´ 1,5 , F. Carneiro3,4,6 , M.B. Barbosa1,2 , D. Nascimento1 , P. Pinto do O M.J. Oliveira1,3 . 1 INEB, Institute of Biomedical Engineering, Oporto, Portugal, 2 ICBAS-Abel Salazar Institute for Biomedical Sciences, Porto, Portugal, 3 Department of Pathology and Oncology from Faculty of Medicine, ˜ Joao ˜ Hospital Porto, Portugal, 4 Department of Anatomic Pathology, Sao Center, Porto, Portugal, 5 FEUP-Faculty of Engineering, Porto, Portugal, 6 IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, Portugal Introduction: Tumours are highly complex microecosystems composed of cancer cells, extracellular matrix (ECM) components and other cell types. The molecular crosstalks established between cancer cells and the surrounding environment are crucial for tumour progression. Macrophages have been described as key elements in this process, preventing the spreading of cancer cells − M1 macrophages − or supporting tumour progression − M2 macrophages. We are particularly interested to elucidate how the ECM and tumour cells contribute to macrophage polarization. Therefore, we are creating a 3D-organotypic culture mode, by decellularizing human colorectal cancer (CRC) tissue fragments and by repopulating them with monocytes and or/tumour cells, mimicking more closely the natural tumour microecosystem. Materials and Methods: We optimized the decellularization protocol and accessed its efficiency as well as tissue morphology and architecture by light microscopy and Scanning Electron Microscopy (SEM). The effect of tissue decellularization on DNA and Glycosaminoglycans (GAGs) contents were evaluated. These matrices were then repopulated with freshly isolated monocytes and allowed to differentiate for 7 days. Additionally, we characterized through immunohistochemistry (IHC) analysis, CD68, CD163, HLA-DR and calprotectin expression profile of macrophage populations in CRC cases from Sao ˜ Joao ˜ Hospital Center. Results and Discussion: Deoxyribonucleic acid (DNA) quantification and 4 ,6-diamidino-2-phenylindole (DAPI) staining confirmed the efficiency of the decellularization method. SEM analysis allowed the visualization of the ECM fiber meshwork while staining with Hematoxylin-Eosin (HE) and Masson’s Trichrome revealed that decellularized fragments retain the histological features of the tissues. Decellularization reduced significantly the GAGS content in normal and tumours but other ECM components, such as laminin or fibronectin, are retained. Preliminary results clearly evidenced that monocytes are able to colonize decellularized matrices and to differentiate into macrophages within the fiber network. Conclusion: The decellularization protocol is effective in decellularizing normal and tumour fragments while retaining, at least partially, tissue architecture and composition. Preliminary results on matrices repopulations suggest that this could be a helpful model to study tumour complexity. At completion of this project we expect to have elucidated the role of tumour cells and of ECM components, derived from the tumour microecosystem, on macrophage differentiation and polarization, contributing to the design of novel therapeutic strategies targeting macrophages. No conflict of interest.