570 MiR-124 Targets Androgen Receptor and Inhibits Tumorigenesis of Prostate Cancer Cells

Poster Session – New Molecular Targets 570 POSTER MiR-124 Targets Androgen Receptor and Inhibits Tumorigenesis of Prostate Cancer Cells R. de Vere White1 , X.B. Shi1 . 1 University of California, Comprehensive Cancer Center, Sacramento, USA Background: The human genome may encode >1000 miRNAs which negatively regulate approximately 60% protein-coding genes. Studies have shown miR-124 is significantly decreased in several cancer types and dysregulation of miR-124 may be involved in the pathogenesis of human cancer. Whether miR-124 contributes to the initiation and progression of prostate cancer (CaP) remains poorly understood. This study was meant to explore the role of miR-124 in CaP. Methods: Total RNA was isolated from fresh-frozen prostatic tissues or cell lines using TRIzol reagent. The miR-124 level was measured using qPCR and/or Northern blot analysis. Since the 3 UTR of androgen receptor (AR) mRNA contains a broadly conserved miR-124-binding site, Western blotting and luciferase assay were performed to validate miR-124-mediated regulation of AR. In addition, the effects of miR-124 on growth of CaP cells were evaluated by WST proliferation assay and tumorigenesis in nude mice. Results: Seven prostate cell lines (2 benign, 5 malignant) and 18 matched benign/malignant prostatic tissues were examined for their miR-124 levels. A reduced expression of miR-124 was seen in all malignant cell lines compared to that in benign cell lines, and in 15 of 18 (83%) CaP samples relative to benign matches. Northern blot analysis of seven cell lines and five matched prostate tissues exhibited similar alteration of miR-124. These data provide strong evidence that miR-124 is significantly reduced in CaP cell lines and in a majority of clinical CaP samples. To validate regulation of AR by miR-124, CaP cells were transiently transfected with synthetic miR124 mimic (miR-124m). We observed miR-124-mediated downregulation of the AR protein and its downsteam effectors PSA and miR-125b. To verify that the miR-124 binding site is responsible for regulation by miR124, the AR 3’ UTR was cloned into the pRIM Reporter-luciferase vector and then cotransfected with miR-124m into CaP cells. Transfection of miR124m resulted in 40% reduction of the luciferase activity. We also examined whether clinical CaP samples having low level of miR-124 overexpress the AR using IHC analysis, and found a reverse correlation between miR-124 and AR expression levels in 7 of 8 CaP samples. These data suggest that miR-124 targets the AR and down-regulation of miR-124 results in increased expression of the AR in CaP cells. We also tested and found that treatment with miR-124m induced significant inhibition of cell proliferation compared to treatment with miR-NC control. We also evaluated the effect of miR-124 on tumorigenesis of 22Rv1 CaP cells and observed that tumors derived from miR-124-transfected cells grew substantially slowly, when compared to miR-NC tumors. Conclusions: Loss of miR-124 expression may be a common event in CaP. Since miR-124 directly targets the AR, downregulation of miR-124 reduces negative regulation, which results in increased expression of the AR in clinical CaP tissue. Additionally, downregulation of miR-124 may contribute to pathogenesis of CaP. 571 POSTER Targeting the GLUT1 Transporter as a Novel Therapeutic Approach to Exploit the Dependency of Cancer Cells On the “Warburg Effect” D.A. Chan1 , P. Pearson2 , J.M. Vernier3 , S. Hershenson4 , J. Freddo5 , R. Tabibiazar6 , A.J. Giaccia7 , P.M. O’Connor8 . 1 UCSF, Department of Radiation Oncology, San Francisco California, USA; 2 Ruga Corporation, Preclinical Development, Palo Alto California, USA; 3 Ruga Corporation, Chemistry, Palo Alto California, USA; 4 Ruga Corporation, Pharmaceutics, Palo Alto California, USA; 5 Ruga Corporation, Clinical Development, Palo Alto California, USA; 6 Ruga Corporation, Palo Alto California, USA; 7 Stanford University School of Medicine, Department of Radiation Oncology, Stanford California, USA; 8 Ruga Corporation, Research & Development, Palo Alto California, USA Tumor metabolism is undergoing a renaissance driven in large-part by identification of ‘genetic drivers’ (e.g., VHL, PTEN, RAS or PI3K) that reprogram tumors to become dependent on glycolysis for growth and survival. Within the repertoire of metabolic changes induced during tumor evolution is the selective up-regulation of a highly facilitative glucose transporter, GLUT1 that feeds the insatiable appetite tumors have for glucose. The dependency of tumors on glucose supplied through a GLUT1-dependent pipeline provides a potential Achilles heel for therapeutic intervention. To identify novel agents capable of selectively killing GLUT1-positive cancer cells we performed a synthetic lethal screen comprising of an isogenic pair of kidney cancer cell lines differing in VHL tumor suppressor status. Interrogation with a 130,000 chemical diversity set derived several lead molecules including STF-31 that selectively killed VHL-deficient, GLUT1 positive cells (Chan et al., Sci Transl. Med., 2011). Expanding beyond these earlier studies we demonstrate the exquisite in vitro and in vivo sensitivity of

Friday 9 November 2012 175 a panel of ovarian cancer cell lines to GLUT1 inhibition including the finding of equivalent activity in cisplatin-sensitive and resistant cell backgrounds (A2780 IC50 = 3.0 nM, A2780/CP IC50 = 3.3 nM). Furthermore, in vivo antitumor studies in orthotopic ovarian cancer models revealed a dosedependent inhibition of tumor growth at well-tolerated doses. Furthermore, efficacy correlated with a drug-induced reduction in fluorodeoxyglucoseuptake by PET imaging compared to vehicle controls. These single-agent and on-going drug-combination studies will be presented and provides a data platform from which to pursue clinical studies targeting patients with GLUT1-dependent tumors. Giaccia, A.J. and O’Connor, P.M. are Joint Senior Authors. 572 POSTER Integrated Genomic Analysis Identifies Candidate Genes in Luminal B Breast Cancer L. Addou-Klouche1 , S. Moulessehoul2 , D. Birnbaum3 , M. Chaffanet3 . 1 Institut Paoli-almettes, Molecular Oncology, Marseille, France; 2 ` Algeria; 3 Institut Paoli-Calmettes, UDL-Sba, Biology, Sidi-Bel-Abbes, Molecular Oncology, Marseille, France Background: Breast cancer is a complex and heterogeneous disease. Characterization of genomic alterations such as amplification of oncogenes and loss of tumor suppressor genes (TSG) combined with expression data could identify candidate genes. We focused on luminal B breast cancer molecular subtype whose clinical course is particularly pejorative and for which no targeted therapy exists. Material and Methods: High-Throughput molecular analysis such comparative genomic hybridization on microarrays (aCGH) was used to characterize the regions targeted by chromosomal alterations in breast cancers. In addition, an integrated analysis of genomic and expression profiling from DNA microarrays has contributed to the identification of candidate genes. Results: We demonstrate that the candidate gene L3MBTL4 is targeted by multiple genomic alterations, suggesting its involvement as a potential TSG in luminal B breast cancer molecular subtype. Furthermore, our comparative analyses of integrated profiles of breast cancers identified specific luminal B molecular subtype candidate genes. Conclusion: High-Throughput molecular analysis of breast cancer has already revealed some part of their potential. Such integrated approaches could contribute to better understand the various levels of the molecular changes in the mammary oncogenesis and identify new markers. 573 POSTER Sonic Hedgehog Pathway Activation Reveals Hexokinase-2-mediated Aerobic Glycolysis as a Novel Target for Medulloblastoma Therapy T. Gershon1 , A. Crowther1 , A. Tikunov2 , I. Garcia1 , H. Yuan3 , J. Macdonald2 , M. Deshmukh4 . 1 UNC-Chapel Hill, Neurology, Chapel Hill NC, USA; 2 UNC-Chapel Hill, Biomedical Engineering, Chapel Hill NC, USA; 3 UNC-Chapel Hill, Radiology, Chapel Hill NC, USA; 4 UNC-Chapel Hill, Cell and Developmental Biology, Chapel Hill NC, USA Medulloblastoma is the most common malignant brain tumor in children. Conventional therapy for medulloblastoma fails to cure many patients and leaves many survivors with long-term neuro-cognitive injury. For children with recurrent medulloblastoma, there is no curative therapy. Targeted therapies offer the possibility of reducing treatment toxicity while improving efficacy. The molecular heterogeneity of medulloblastoma, however, may complicate the selection of appropriate targets. To define molecular targets for therapy that may be valid across medulloblastoma subgroups, we probed for distinguishing metabolic features common to medulloblastoma and cerebellar granule neuron progenitors (CGNPs), putative cells of origin from which medulloblastomas arise. We performed an integrated analysis of metabolism and gene expression in CGNPs with and without Sonic Hedgehog (Shh), their endogenous mitogen. Because our analysis highlighted Hexokinase-2 (Hk2) as key metabolic regulator induced by Shh, we studied the effect of conditional genetic Hk2 deletion in CGNP development. We then we crossed Hk2 conditional knockout mice with transgenic SmoM2 mice that develop spontaneous medulloblastoma and determined changes in SmoM2-driven tumorigenesis. We found Shh induced aerobic glycolysis by driving Hk2 expression in CGNPs, both in vitro and in vivo. Hk2 deletion abrogated Shhinduced glycolysis and altered the strict regulation of CGNP differentiation in development. Medulloblastomas retained Hk2 expression typical of CGNPs. While all SmoM2 mice developed medulloblastoma, deletion of Hk2 markedly reduced tumor malignancy and increased survival. While 100% of SmoM2 mice died of medulloblastoma by P20, SmoM2 mice with Hk2 deletion survived a median of 31 days and 30% were longterm survivors. Comparison of pathology of medulloblastoma with and without intact Hk2 demonstrated that Hk2 deletion reduced tumor growth specifically by promoting differentiation.