ESTRO 36 Abstract Book

S200 ESTRO 36 _______________________________________________________________________________________________

Material and Methods To predict the potential targets of miR-200c, a microRNA database was used for bioinformatics analysis. Malignant glioma (U251), breast cancer (SKBR3, MDAMB468) and lung carcinoma (A549) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were transfected with pre-miR-200c or control pre-miRNA using siPORTNeoFX™ transfection reagent (Ambion, Austin, TX, USA). Anti-miR-200c was mixed with Opti-MEM (Invitrogen, Grand Island, NY, USA), incubated and added directly to cells. RT-PCR was performed using the Taqman miRNA reverse transcription kit and the Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Clonogenic assay, immunoblotting and immunocytochemisty was perfomed. Results Ectopic overexpression of miR-200c led to down- regulation of p-AKT, p-EGFR, and p-HER2 and increased the radiosensitivity of U251, A549, SKBR3, and MDA-MB- 468 cells. In contrast, a miR-200c inhibitor led to up- regulation of p-AKT, p-EGFR, and p-HER2 and decreased radiation-induced cell killing. miR-200c led to persistent γH2AX foci formation and down-regulated pDNA-PKcs expression. Autophagy and apoptosis were major modes of cell death. Bioinformatics analysis predicted that miR-200c could have association with EGFR, AKT, MAPK, VEGFA, HIF1AN. We also confirmed that miR-200c downregulated expression of VEGF, HIF-1α, and MMP2. Overexpression of miR-200c inhibited invasion, migration, and vascular tube formation. These were associated with downregulation of E-cadherin and EphA2, and up-regulation of N-cadherin. miR-200c showed no observable cytotoxic effect on normal human fibroblasts and normal human astrocytes. Conclusion Taken together, our data suggest that miR-200c is an attractive target for improving the efficacy of radiotherapy via unique modulation of the complex regulatory network controlling cancer pro-survival signaling and EMT. PV-0371 Novel molecular radiobiology for personalised prostate cancer radiotherapy N. McDermott 1 , A. Meunier 1 , T. Jameson 2 , A. Mansour 2 , C. Haynes 2 , A. Flores 2 , A. O'Callaghan 1 , L. Marignol 1 1 Trinity Centre for Health Sciences Discipline of Radiation Therapy, Radiation Therapy, Dublin, Ireland 2 Mount Sinai School of Medicine, International Health, New York, USA Purpose or Objective The integration of tumour-specific biological parameters to the decision-making process is anticipated to overcome the recognised limitations of the current risk stratification system for prostate cancer and transform the practice of radiation oncology. Molecular imaging techniques are rapidly advancing our ability to assess the extent and aggressiveness of prostate cancer. Differential analyses of extensive genetic profiles of specimens have progressed the use of genetic signatures from tumour tissue in providing additional prognostic information. Adopting an hypothesis-based approach to the identification of novel radiobiology that can assist the personalisation of prostate radiotherapy, we propose that that the signalling pathways that regulate several cancer hallmarks and are responsive to hypoxia, such as Notch and YB-1 regulate the molecular response of cells to radiation. Material and Methods The analysis of a panel of 22Rv1 prostate cancer cells was used to support the identification of novel biomarkers of radioresistance. First, an isogenic model of radioresistance was generated in 22Rv1 prostate cancer cells through exposure to 30 x 2-Gy dose fractions. Second, radioresistance was induced in 22Rv1 cells through exposure to hypoxic conditions (0.5% O2, 24hrs). miRNA

profiling of these samples was performed and validated by RT-PCR. Novel signalling interactions were characterised by western blotting, and a series of cell-based assays. Results Exposure to fractionated 2Gy-irradition to a cumulative total dose of 60Gy selected for 22Rv1 cells increase in clonogenic survival following irradiation (1.3 fold increase in survival after 2Gy and 2.2 fold increase after 10Gy) when compared to both parent 22Rv1 and aged-matched controls. The cross-evaluation strategy of the molecular modifications associated with a combination of radiobiological factors has identified miR-4284 as down regulated amongst radiation resistant models. Further evaluation of this miRNA indicates interaction with RLIM, RASGEF1, YB-1 and Notch-3. YB-1 inhibition with Fisetin significantly reduced clonogenic survival following irradiation, and modified Notch-3 receptor activation. Analysis of RNA extracted from a series of pooled samples from prostate cancer patients identified elevation of notch-3 mRNA levels in higher grade and hypoxic tumours. Validation in cell lines further identifies modification in Notch-3 activation following 5-Gy irradiation. Conclusion This study identifies novel molecular radiobiology that may explain the multiple effects of radiation on the molecular biology of prostate cancer cells. This work has the potential to influence future direction of suitability and treatment of radiotherapy prostate patients. PV-0372 Histology-specific quantitative mapping and targeting of glucose and glutamine metabolism in NSCLC T.W.H. Meijer 1 , P.N. Span 1 , W.J.M. Peeters 1 , R. Biemans 3 , L.F. De Geus-Oei 2 , D. Vriens 2 , L.J. Dubois 3 , J. Bussink 1 1 UMC St Radboud Nijmegen, Radiation Oncology, Nijmegen, The Netherlands 2 Leiden university medical center, Radiology, Leiden, The Netherlands 3 Maastricht University Medical Centre, Radiation Oncology, Maastricht, The Netherlands Purpose or Objective Increased glycolysis and glutamine use are related to resistance to radiotherapy. Therefore, targeting tumor cell metabolism may improve radiotherapy efficacy in NSCLC. In this prospective cohort study, we describe pharmacokinetic rate constants of 18 F-FDG metabolism (K 1 -k 3 ) and fractional blood volume (V B ) in regions with different levels of glucose metabolic rate (MR glc ) and compare these between the major NSCLC histological subtypes (adeno- (AC) and squamous cell carcinomas (SCC)). Furthermore, glycolytic rate and growth delay plus apoptotic index by glucose and/or glutamine inhibition were assessed in six NSCLC cell lines in vitro. Material and Methods One-hour dynamic 18 F-FDG-PET/CTs were acquired in 38 NSCLC patients (tumor size at least 30 mm in diameter). Parametric images of Patlak MR glc values were obtained. Lesions were delineated using the fuzzy locally adapted Bayesian (FLAB) algorithm. Tumors were divided into three equal volumes of increasing MR glc , in which K 1 -k 3 and V B were computed. For in vitro experiments, AC (H522, HCC827, H1975) and SCC (H520, H226, SW900) NSCLC cell lines were used. Glycolytic rate of cell lines was assessed by the percentage extracellular acidification rate (% ECAR) under normoxia and physiologic amount of glucose (i.e. 1.5 mM) using Seahorse. Growth delay and apoptosis analyses were performed under normoxia and 1.5 mM glucose using IncuCyte. To examine the effect of metabolic inhibition on growth delay and apoptotic index, the glycolysis inhibitor lonidamine and/or glutaminase inhibitor 968 were used.