european journal of cancer 48, suppl. 5 (2012) S25–S288
241 Tumor-derived Granulocyte-macrophage Colony Stimulating Factor is Responsible for Accumulation of Pro-invasive Microglia/macrophages and Glioma Progression M. Sielska1 , P. Przanowski1 , J. Kucharska2 , K. Gabrusiewicz1 , M. Kijewska1 , M. Maleszewska1 , M. Zawadzka1 , B. Kaminska1 . 1 Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland, 2 Nencki Institute of Experimental Biology, Laboratory of Confocal Microscopy, Warsaw, Poland Introduction: Experimental and clinical studies show an important role of glioma-inﬁltrating macrophages in glioma progression. Macrophages are attracted by tumor-released molecules and instead of initiating antitumor responses, those cells support invasion, angiogenesis, extracellular matrix remodeling and immunosuppression indifferent types of tumors. In some experimental cancer, M-CSF/CSF-1 (macrophagecolony-stimulating factor/colony-stimulating factor 1) was reported as a crucial factor regulating inﬁltration and function of tumor-associated macrophages. We found that GL261 glioma cells express a marginal amount of csf-1 mRNA but the expression of csf-2/gm-csf (colony-stimulating factor 2/ granulocyte macrophagecolonystimulating factor) is high in comparison to non-transformed astrocytes. Material and Method: We generated EGFP-GL261glioma cells stably expressing GM-CSF speciﬁc or control shRNA. Silencing of GM-CSFexpression was conﬁrmed by qPCR and ELISA. Intracranial growth of control and GM-CSF depleted glioma cells, macrophage inﬁltration (Iba1 staining and determination of CD11b+ CD45high and CD11b+ CD45low populations in tumor bearing brains by ﬂow cytometry), angiogenesis (vWFstaining) and animal survival were evaluated. Osteopetrotic mice (op/op)deﬁcient in M-CSF were used to evaluate a role of M-CSF in glioma progression. Results and Discussion: Silencing of gm-csf expression did not affect basal proliferationor survival of cultured glioma cells. Intracranial gliomas depleted of GM-CSF showed highly reduced accumulation of Iba-1+ cells and signiﬁcantly diminished tumor invasion and angiogenesis in comparison to control gliomas. Mice implanted with GM-CSF depleted glioma cells survived longer than mice with control gliomas. Interestingly, the growth of GL261 gliomas and accumulation of macrophages/microglia were not affected in osteopetrotic (op/op) mice, deﬁcient inM-CSF. The expression of CSF-2 (but not CSF-1) was highly up-regulated in glioblastoma multiforme patients. Kaplan–Meier survival curves acquired from the Rembrandt depository showed inverse correlation between CSF-2 gene expression and survival of glioma patients. Our results demonstrate that glioma-derived GM-CSF controls recruitment of inﬁltrating microglia/macrophages and contributes to glioma progression. Conclusion: GM-CSF is responsible for tumor-driven accumulation of brain resident, peripheral macrophages and may be a new target for glioblastoma therapy. 242 Identiﬁcation of EPHA3 as a Candidate Tumor Suppressor in a Screen for Cellular Senescence J. Lahtela1 , T.L. Hunsaker2 , M.J. Brauer2 , J. Lee2 , A. Hemmes1 , S. Koopal1 , L. Corson2 , P.K. Jackson2 , E.W. Verschuren1 . 1 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland, 2 Genentech Inc., Department of Cell Regulation, South San Francisco, USA Introduction: Premature engagement of a cellular senescence program is a common cellular response to prolonged oncogene activation or loss of tumor suppressor function, acting as a physiological tumor suppression mechanism in premalignant human tumors. Characteristic features of cellular senescence comprise a permanent cell cycle arrest associated with cell morphological changes, such as cellular and nuclear ﬂattening, and an increase in senescence-associated b-galactosidase (SA-b-Gal) activity. Senescence is also commonly triggered by activation of the (Ets/)p16INK4a /Rb tumor suppressor pathway. Material and Methods: Since oncogene-induced senescence (OIS) is a commonly detected in cells containing intact cellular DDR and p16INK4a pathways, we reasoned that a siRNA screen in untransformed cells should identify putative tumor suppressor genes. We developed a screen applying cell morphology and proliferation parameters to identify senescence-inducing kinome siRNAs, expecting to identify putative tumor suppressors. Results and Discussion: We successfully identiﬁed twelve kinases as novel regulators of cellular senescence. One such candidate was the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human lung adenocarcinomas and colorectal cancers. We show that senescence in response to loss of EPHA3 expression is partially conferred by known actors of cellular senescence p16INK4a and p53. Structural analysis of intracellular EPHA3 tumor-associated point mutations suggests molecular explanations on how mutations may impact on receptor kinase activity. To functionally show the consequence of somatic mutations, we express near-endogenous levels of GFP-tagged EphA3 variants, which are correctly membrane localised and internalised upon ligand treatment. Importantly, we show that selected kinase domain point mutations cause a decrease in receptor expression level and/or normalised receptor tyrosine kinase (RTK) activity.
Conclusions: Our study describes a new strategy to uncover candidate tumor suppressors, and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing tumor suppressor pathways are inactive. 243 Triple Negative Breast Carcinoma Cells Directly Contribute to Tumor Vasculature by Endothelial Differentiation Capability I. Plantamura1 , M.V. Iorio1 , M. Tortoreto1 , T. Triulzi1 , P. Casalini1 , M. Sasso1 , M. Campiglio1 , C. Tripodo2 , A. Balsari3 , E. Tagliabue1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Experimental Oncology, Milan, Italy, 2 Universita` degli Studi di Palermo, Department of Health Sciences, Palermo, Italy, 3 Universita` degli Studi di Milano, Institute of Human Morphology and Biomedical Sciences “Citta` Studi”, Milan, Italy Introduction: Very aggressive at a clinical point of view, Triple Negative Breast Cancers (TNBCs) represent a question mark in BC biology. Here we explored TNBC vascular properties, particularly focusing on the capability of this subgroup of BC to create vascular structures. Material and Method: Co-staining of FFPE (Formalin-Fixed Parafﬁn Embedded) human TNBC specimens with anti-pan epithelial cytokeratins or anti-p53, and anti-CD31 or CD34 was evaluated by Immunoﬂuorescence. Endothelial-like functional properties were investigated in vitro in BC cell lines seeded on Matrigel by Tube formation assay and in vivo by Confocal microscopy on frozen tumor sections. In vitro Vasculogenic mimicry (VM) and in vivo tumor growth were carried out impairing the main pathways involved in vessel formation by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or siRNA-mediating silencing. Results and Discussion: Immunoﬂuorescence analysis indicated that only TNBCs show an endothelial-like phenotype. Moreover, analysis of VM revealed vascular channel formation in all TNBC cell lines, whereas no vascular structures were generated by luminal or HER2-positive cells. In vitro VM also reﬂected a property of TNBC cells in vivo, where vascular lacunae were identiﬁed only in TNBC-derived tumors in comparison with a luminal xenograft model. To elucidate the mechanisms involved in the VM capability of TN tumor cells lines, we analyzed their VM in presence of Sunitinib, targeting VEGFR, PDGFR and FGFR, or Bevacizumab, against VEGFA. Whereas treatment with Sunitinib completely abrogated the formation of vascular channels in vitro in all TNBC cell lines, the slight effect exerted by Bevacizumab suggested that this property is related to a VEGF-independent mechanism. In keeping with this evidence, siRNA-mediating silencing of the single receptors targeted by Sunitinib revealed a crucial role exerted by FGFR2 and PDGFR-b-mediated networks. Consistent with in vitro data, Sunitinib induced tumor regression in TNBC xenograft models, whereas only a slight effect on tumor size was observed upon Bevacizumab treatment. Conclusion: TNBC cells display an endothelial-like phenotype. This phenomenon is likely to be responsible of the aggressive nature of this BC subtype and the increased sensitivity to Sunitinib seems to rely on the speciﬁc impairment of FGFR2 and PDGFR-b-mediated pathways, new promising speciﬁc therapeutic targets. 244 Intratumoral Societies − Subpopulations of Tumor Cells Expressing Different Levels of ADAM23 Seems Responsible for Different Aspects of Malignancy G.F. Barnabe1 , E.T. Costa1 , P.F. Asprino1 , M.H. Nagai2 , B. Malnic2 , R. Chammas3 , A.A. Camargo1 . 1 Ludwig Institute for Cancer Research, ˜ Paulo, Molecular Oncology Center, Sao Paulo, Brazil, 2 University of Sao ˜ Paulo Department of Biochemistry, Sao Paulo, Brazil, 3 University of Sao Medical School, Department of Radiology, Sao Paulo, Brazil Background: Cancer evolve through the accumulation of genomic and epigenomic alterations that enable tumor progression towards malignancy. Epigenetic silencing of ADAM23 (A Desintegrin And Metalloprotease domain 23) gene is a recurrent event in a wide range of tumors, including pancreatic, breast, gastric and colorectal tumors. Hypermethylation of ADAM23 gene promoter has been associated with metastatic disease and poor overall survival in breast cancer patients (Verbisck et al. 2009). However the precise biological effects of ADAM23 downregulation during tumor progression is unknown. Here we sought to further clarify the role of ADAM23 in tumor progression through functional characterization of ADAM23 in vivo and in vitro. Material and Methods: We stably knocked down ADAM23 expression in MDA-MB-435 tumor cells using shRNA and ectopically expressed human ADAM23 in CMS5a, a mouse ﬁbrosarcoma cell line. Cells transfected with empty vectors were used as controls in all comparisons. Invasion assays were performed embedding cell spheroids in a tri-dimensional collagen-type I matrix. Soft-agar and tumorigenesis assays were performed according to standard protocols. ADAM23 expression in primary tumors displaying ADAM23 promoter hypermethylation was analyzed by in situ hybridization (approved ethics committee).