S86 EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242

surrounding tissue, indicating that the polarity protein Par3 counteracts diseaseprogression through control of proper tissue integrity.Conclusion: We identified crucial roles of conserved regulators of polarity inkeratinocyte proliferation and apoptosis, and demonstrated that impaired Par3function affects skin tumor onset and progression. We are further investigatinghow the cortical Par3 complex links cell polarity with key oncogenic signalingpathways driving non-melanoma skin cancers. These studies will help toprovide valuable insight into the molecular coupling of cyto-architecture andtissue homeostasis by polarity proteins − findings that may prove important toprevent formation and progression of cancer.No conflict of interest.

360 Reduced promoter methylation and increased expression ofCSPG4 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. 1University Hospital Heidelberg, Department ofOtorhinolaryngology Head and Neck Surgery, Heidelberg, Germany,2German Cancer Research Center, Division of Epigenomics and CancerRisk Factors, Heidelberg, Germany, 3University Hospital Heidelberg,Division of Experimental Neurosurgery Department of Neurosurgery,Heidelberg, Germany, 4National Center for Tumor Diseases, NCT TissueBank, Heidelberg, Germany, 5University Hospital Heidelberg, Department ofMedicine V, Heidelberg, Germany

Background: HNSCC biology is strongly influenced by various geneticalterations that appear in early tumor stages. Moreover there is now increasingevidence that epigenetic changes like DNA methylation also contributesignificantly to malignant transformation. The expression level of markersof undifferentiated cells might be accurate prognostic indicators in cancer.Interestingly, there are hints that chondroitin sulfate proteoglycan 4 (CSPG4) isexpressed on normal neural stem cells and cancer stem cells of gliomas andits 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 CSPG4expression in a panel of HNSCC tumors and normal controls. Therefore weused immunohistochemical staining, qPCR and in silico expression analysis ofpublic available expression microarray datasets. The DNA-methylation statusof a CpG-Island in CSPG4 promoter in HNSCC tumors was analyzed by thehighly innovative MassARRAY technique and validated by 5-AZA treatment oftwo HNSCC-cell lines. Finally the CSPG4 expression data and the methylationstatus of the CSPG4 promoter were correlated to the survival prognosis of theHNSCC patients.Results: The expression analysis revealed a dramatic CSPG4 mRNAoverexpression in HNSCC tumors and an increased CSPG4 protein expressionin a subgroup of HNSCCs. Next we identified hypomethylation of the CSPG4promoter in HNSCC which correlated with high CSPG4 mRNA and proteinexpression. This was confirmed by 5-AZA treatment of two HNSCC celllines.Finally we found that high CSPG4 expression and low promoter methylationare significantly associated with an adverse progression-free and overallsurvival.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 linkedto tumor CSPG4 promoter methylation and protein expression. We foundthat CSPG4 expression is often dramatically increased in HNSCC tumors,a result consistent with our detection of generally lower CSPG4 promotermethylation. These data suggest that in HNSCC tumor cells, demethylation atthe CSPG4 promoter contributes to increased CSPG4 expression. In supportof this possibility, we found that treating HNSCC cells harboring a highlymethylated CSPG4 promoter with demethylation agents caused CSPG4 re-expression.In conclusion our findings support our initial hypothesis that CSPG4 is animportant player in HNSCC biology.No conflict of interest.

361 The putative tumor suppressor gene Dickkopf-3 (DKK3) andits role in the carcinogenesis of breast cancer

E. Lorsy1, J. Veeck1, R. Knuchel1, E. Dahl1. 1University Hospital RWTHAachen, Pathology, Aachen, Germany

Introduction: The expression of the putative WNT signaling inhibitorDickkopf-3 (DKK3) is downregulated in several tumor entities. Previously, wehave shown that downregulation of DKK3 in human breast cancer is due toDKK3 promoter hypermethylation and that this molecular lesion is associatedwith unfavorable patient prognosis. In our current study, we are analyzing thebiological function of DKK3 in human breast cancer cell lines.Materials and Methods: MDA-MB-231 and MDA-MB-436 basal-type as wellas MCF7 and MDA-MB-453 luminal-type breast cancer cell lines, all with noendogenous DKK3 expression, were stably transfected using a fulllength DKK3

cDNA containing vector or empty vector. In vitro cell culture assays determinedthe impact on proliferation, colony formation and substrate adhesion.Results: Real-time PCR and Western blot analysis confirmed abundant re-expression of DKK3 mRNA and protein in DKK3-transfected MDA-MB-231,MDA-MB-436, MDA-MB-453 and MCF7 cells. MDA-MB-436 cells switchedfrom a mesenchymal to a more epithelial morphology after transfectionwith DKK3 accompanied with increased expression of epithilial markers(e.g. Claudin 1 and ZO-1). DKK3 clones of both the luminal and thebasal-type exhibited reduced growth capabilities in comparison with mockclones (p = 0.002 for MDA-MB-436 and p = 0.01 for MCF7). DKK3-clonesof the basal-type cell line MDA-MB-436 attached significantly faster to BDMatrigel™ (Basement Membrane Matrix) than mock clones (p < 0.0001), whileno differences were observed in luminal-type MCF7 cells (p > 0.05). Thisdiscrepancy will be further addressed with additional cell lines, as functionalanalyses with other cell lines and assays (e.g. apoptosis) are still ongoing.Conclusions: DKK3 may represent a novel tumor suppressor gene in normalbreast tissue since DKK3 is capable of suppressing cell proliferation of humanbreast cancer cells in vitro and DKK3 promoter methylation in human tumorsis associated with poor survival. Ongoing studies are analyzing the influenceof DKK3 on apoptosis and invasion in vitro. In addition, in vivo experimentsusing dkk3-knockout mice will be performed.No conflict of interest.

362 BMP4 suppresses breast cancer metastasis throughdown-regulation of G-CSF

R.L. Anderson1, Y. Cao1, A. Swierczak1, B.L. Eckhardt2, J.A. Hamilton3.1Peter MacCallum Cancer Centre, Research Division, East MelbourneVictoria, Australia, 2MD Anderson Cancer Center, Department of BreastMedical Oncology, Houston Texas, USA, 3The University of Melbourne,Department of Medicine, Melbourne Victoria, Australia

Background: Most deaths from breast cancer are due to the onset ofmetastatic disease, for which there are few curative therapies. A betterunderstanding of molecular mechanisms driving metastasis offers thepossibility of improved therapies. In our screens for metastasis regulators,we discovered that bone morphogenetic protein 4 (BMP4) is a powerfulmetastasis suppressor. BMP4 belongs to the transforming growth factor(TGF-b) superfamily and is an important cytokine regulating many biologicalfunctions in a context dependent manner.Materials and Methods: We have used preclinical models of spontaneousbreast cancer metastasis to explore the mechanisms by which BMP4 regulatesmetastasis. We have also investigated the prognostic significance of BMP4 intissue arrays of cancers from breast cancer patients.Results: High levels of BMP4 correlated with improved outcome in breastcancer patients. In our preclinical models, BMP4 did not alter primarytumour growth but profoundly reduced development of metastases. In therapymode, recombinant BMP4 (rBMP4) prolonged survival of mice bearing highlymetastatic mammary tumours. In seeking the mechanism by which BMP4suppresses metastasis, we found that either expression of BMP4 in mammarytumour cells in vitro or treatment with rBMP4 led to down-regulation of G-CSFsecretion.An analysis of plasma from mice bearing metastatic mammary tumoursrevealed high levels of G-CSF that were reduced in mice whose tumoursexpressed BMP4. In parallel with reduced G-CSF, we found a lower proportionof myeloid derived suppressor cells (MDSC) in peripheral blood. Directinjection of G-CSF into mice stimulated the mobilisation of MDSC andenhanced the metastatic capacity of mammary tumours. The MDSC inducedby treatment of mice with G-CSF were able to suppress CD4+/CD8+ T cellproliferation, demonstrating their immunosuppressive activity. In contrast, theMDSC present in mice bearing BMP4 expressing tumours had reducedimmunosuppressive activity. We have demonstrated that BMP4 regulatesG-CSF expression through inhibition of NFkB signaling.A direct impact of G-CSF signaling on metastasis was demonstrated byshowing that treatment of tumour-bearing mice with a neutralising antibodyagainst G-CSF receptor (G-CSFR) led to a reduction in MDSC and a reductionin spontaneous metastasis of mammary tumours to lung and bone.Conclusions: These observations open a path for a new therapy for advancedbreast cancer based on activation of BMP4 signaling and/or blockade ofGCSFR signaling.No conflict of interest.

363 The ING1 tumour suppressor induces senescence via alteringendocytosis

K. Riabowol1, U.K. Rajarajacholan1, S. Thaalippilly1. 1University of Calgary,Biochemistry & Molecular Biology and Oncology, Calgary Alberta, Canada

Background: The INhibitor of Growth (ING) proteins act as type II tumorsuppressors and epigenetic regulators, being stoichiometric members ofhistone acetyltransferase and histone deacetylase complexes. Alternative