Poster Sessions european journal of cancer 48, suppl. 5 (2012) S25–S288 S43 176 Roles of PDCD4 and EIF4A1 in Breast Cancer A. Modelska 1 , J. Le Quesne 1 , A. Bottley 2 , M. Osborne 1 , R. Russell 1 , J. Hadfield 1 , K. Spriggs 2 , P. Pharoah 3 , C. Caldas 1 . 1 Cambridge Research Institute, Cancer Research UK, Cambridge, United Kingdom, 2 University of Nottingham, Centre for Biomolecular Sciences, Nottingham, United Kingdom, 3 University of Cambridge, Oncology, Cambridge, United Kingdom Introduction: Programmed Cell Death 4 (PDCD4) is a tumour suppressor protein that is important in translational control. It binds and inhibits eukaryotic Initiation Factor 4A1 (eIF4A1), a helicase unwinding 5 ′ UTR secondary structures. PDCD4 appears to function in different ways depending on cell type and genetic background and, therefore, it is important to study its role in a context-dependent manner. This project focuses on understanding global and message-specific translation regulation by PDCD4 and eIF4A1, and phenotypes induced by those interactions in breast cancer. Material and Method: Immunohistochemical analysis of PDCD4 and eIF4A1 expression was performed on a set of 3,605 cases of archival breast tumours (SEARCH). Transient knock-downs of PDCD4 and eIF4A1 were obtained using siRNA. Polysome fractionation was performed on sucrose gradients, and total, subpolysomal and polysomal RNA was collected from control, PDCD4 and eIF4A1 knock-down cells, and analysed on gene expression microarray, followed by differential gene expression and pathway enrichment analysis. PDCD4 and eIF4A1 levels were assayed by Western blotting. Stable PDCD4 knock-down and over-expression cell lines were generated using lentiviral infections, and growth and migratory properties were assayed. Results and Discussion: In the patient cohort studied, PDCD4 expression is strongly predictive of survival in estrogen receptor positive breast cancer patients. PDCD4 resides in both nuclear and cytoplasmic compartments. Nuclear PDCD4 correlates negatively with grade, stage, tumour size and lymph node status, and the proliferation marker Ki67. It positively correlates with the Luminal A tumour subtype and Bcl-2. In contrast, eIF4A1 is predictive of poor outcome in estrogen receptor negative tumours. PDCD4 levels differ across breast cancer cell lines and increase with confluency. Transient knock-down of PDCD4 in MCF7 exerts few phenotypic effects, whereas knock-down of eIF4A1 causes morphological changes and decrease in cell growth. Stable PDCD4 over-expression results in slower growth phenotype. Transient PDCD4 knock-down yields very few differentially expressed genes, consistent with the very high levels of eIF4A1 expressed by this cell line. In contrast, eIF4A1 knock-down has a very pronounced effect on the polysome profile and affects many genes involved in cell adhesion, cytoskeleton remodelling, apoptosis, development, cell cycle, translation and metabolism. Conclusion: Here we show that PDCD4 and eIF4A1 can differentially predict survival depending on breast cancer subtype. Moreover, knock-downs of these molecules in MCF7 affect the expression of different sets of genes, while only eIF4A1 knock-down induces an obvious phenotype in cell culture. We propose a model to explain how dysregulation of these key translational regulators contributes to the malignant phenotype in breast cancer. 177 Effect of Androgens on the Expression of Ca2+-binding Protein, Regucalcin, and Ca2+-channels in MCF-7 Cells R. Marques 1 , C.V. Vaz 1 , C.G. Peres 1 , I. Gomes 1 , C.R. Santos 1 , C.J. Maia 1 , S. Socorro 1 . 1 University of Beira Interior, Health Sciences Research Centre, Covilh ˜ a, Portugal Background: Androgens are the precursors of estrogens, but in addition to their role as precursors hormones, have also been suggested as important agents in breast physiology. However, the precise function of androgens on breast cells proliferation and malignancy has not been fully elucidated. Ca 2+ is a ubiquitous second messenger that controls mostly of cell signaling pathways, and altered Ca 2+ homeostasis has been associated with development of breast cancer. RGN is a Ca 2+ -binding protein playing an important role in intracellular Ca 2+ homeostasis. Recently, we demonstrated that RGN is an androgen-target in human prostate LnCap cells. Others have also shown that Ca 2+ -permeable channel TRPM8 was androgen regulated in these cells, and that androgens control the expression and activity of L-type Ca 2+ channels in several types of contractile cells. In the present study we will determine the effect of 5a-dihydrotestosterone (DHT) on the expression of RGN, TRPM8 and L-type Ca 2+ channels in MCF-7cells. Material and Methods: MCF-7 cells were exposed to 0, 1, 10 or 100 nM of DHT for 0, 6, 12 and 24 h. 1 nM DHT experiments were repeated in presence of 1 mM Flutamide, 1 mg/mL ciclohexamide or 100 nM ICI182,780. Expression of RGN, TRPM8 and L-type Ca 2+ -channels in different experimental groups (n = 6) was evaluated by means of real-time PCR and/or Western Blot techniques. Results and Discussion: RGN expression in MCF-7 cells was down- regulated by DHT (1 nM), a similar result to that previously described in LnCap cells. The determination of androgenic effects on the expression of Ca 2+ -channels is underway. DHT concentration is reported to be significantly higher in breast carcinoma tissue that in plasma and androgen receptor (AR) is expressed in the majority of breast cancer specimens. Furthermore, estrogen- and progesterone receptor-negative breast tumours still significantly express AR, which highlights the importance of androgen signalling in breast cancer development. The results presented herein indicate that androgenic actions in breast cells underlie the maintenance of intracellular Ca 2+ levels by controlling the expression of Ca 2+ -homeostasis regulators. Conclusion: RGN, and likely Ca 2+ -channels, expression is under DHT control in MCF-7 cells, suggesting that androgens’ role in breast pathophysiology is linked to the control of intracellular Ca 2+ homeostasis. 178 E6AP Ubiquitin Ligase Regulates PML-induced Senescence in Myc-driven Lymphomagenesis K. Wolyniec 1 , J. Shortt 1 , S. Opat 2 , R.W. Johnstone 1 , C.L. Scott 3 , S. Fox 1 , A. Strasser 3 , S. Haupt 1 , Y. Haupt 1 . 1 Peter MacCallum Cancer Centre, Melbourne Victoria, Australia, 2 Monash Medical Centre, Department of Clinical Haematology, Melbourne Victoria, Australia, 3 Walter and Eliza Hall Institute of Medical Research, Melbourne Victoria, Australia Neoplastic transformation requires the elimination of key tumor suppressors, which may result from E3 ligase-mediated proteasomal degradation. We previously demonstrated a key role for E3 ubiquitin ligase E6AP (E6-associated protein) in the regulation of PML (promyelocytic leukaemia protein) stability and formation of PML nuclear bodies. Here, we report the involvement of the E6AP- PML axis in B-cell lymphoma. A partial loss of E6AP attenuates Myc-induced B-cell lymphomagenesis. This tumor suppressive action is achieved by the induction of cellular senescence. B-cell lymphomas deficient for E6AP express elevated levels of PML and PML-nuclear bodies, and increased expression of senescent markers, including p21, p16 and H3K9me3. Importantly, E6AP expression is elevated in ~60% of human Burkitt lymphomas, and down- regulation of E6AP in B lymphoma cells restores PML expression with a concomitant induction of cellular senescence. Our findings demonstrate that E6AP regulation of PML-induced senescence is essential for B-cell lymphoma progression. This provides a molecular explanation for the down-regulation of PML in Non-Hodgkin lymphomas, thereby suggesting a novel therapeutic approach for restoration of tumor suppression in B-cell lymphoma. 179 Regulation of STEAP1 Expression in Prostate by Sex Steroid Hormones I. Gomes 1 , P. Arinto 1 , C.R.A. Santos 1 , S. Socorro 1 , C. Lopes 2 , C.J. Maia 1 . 1 Health Sciences Faculty − University of Beira Interior, Health Sciences Research Center, Covilh˜ a, Portugal, 2 University of Porto, Institute for the Biomedical Sciences Abel Salazar, Porto, Portugal Introduction: Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is an overexpressed gene in prostate cancer. It is localized in the plasma membrane of epithelial cells, specifically in cell-cell junctions. There are evidences that STEAP1 may be a useful marker for several types of cancer with potential for immunotherapy applications. However, studies involving STEAP1regulation in prostate cells are scarce. It is well known that sex steroid hormones are involved in maintenance of prostate physiology and may promote proliferation of prostate cancer cells. Therefore, the effects of dihydrotestosterone (DHT) and 17b-estradiol (E 2 ) in regulation of STEAP1 were determined. Moreover, it is not well understood the clinical significance of STEAP1 expression in human prostate cancer. Therefore, it is crucial to evaluate whether STEAP1 correlates with clinical data. Material and Methods: In vitro, LNCaP prostate cancer cells were treated with different doses of DHT or E2 for several periods of time. The possible signaling pathways were accessed by exposing LNCaP cells to flutamide, ICI and cycloheximide. In vivo, 3 months old wistar rats were divided into 4 distinct groups that included orchidectomised animals treated with either DHT or vehicle, and intact animals treated with E 2 or vehicle alone. STEAP1 expression in each experimental group was carried out by Real-time PCR and Western blot. Immunohistochemistry was performed in tissue microarrays (TMAs) of human prostate cancer and non-neoplastic lesions. Correlation between STEAP1 immunoreactivity and clinical data was established using a SPSS program. Results and Discussion: STEAP1 expression is inhibited by the presence of DHT and E 2 in LNCaP cells. The effect of DHT is time- and dose-independent, but E 2 appears to trigger STEAP1 down-regulation only after 24h at all concentrations. The signaling pathways by which DHT decrease STEAP1 expression seems to involve the activation of AR. In vivo, castration of adult rats increases STEAP1 protein expression when compared to intact rats, and treatment with DHT abrogates the effect of castration in STEAP1 expression. However, these effects were not observed at STEAP1 mRNA level, suggesting that mechanisms underlying the regulation of translation may be involved. E2- treated intact animals also show lower levels of STEAP1 when compared to intact animals treated with vehicle alone. Semi-quantification of STEAP1 immunoreactivity in prostate cancer and statistical analysis are underway.