Yoko Hasegawa

Fatty Acid Synthase Is a Key Target in Multiple Essential Tumor Functions of Prostate Cancer: Uptake of Radiolabeled Acetate as a Predictor of the Targeted Therapy Outcome

Fatty acid synthase (FASN) expression is elevated in several cancers, and this over-expression is associated with poor prognosis. Inhibitors of FASN, such as orlistat, reportedly show antitumor effects against cancers that over-express FASN, making FASN a promising therapeutic target. However, large variations in FASN expression levels in individual tumors have been observed, and methods to predict FASN-targeted therapy outcome before treatment are required to avoid unnecessary treatment. In addition, how FASN inhibition affects tumor progression remains unclear. Here, we showed the method to predict FASN-targeted therapy outcome using radiolabeled acetate uptake and presented mechanisms of FASN inhibition with human prostate cancer cell lines, to provide the treatment strategy of FASN-targeted therapy. We revealed that tumor uptake of radiolabeled acetate reflected the FASN expression levels and sensitivity to FASN-targeted therapy with orlistat in vitro and in vivo. FASN-targeted therapy was noticeably effective against tumors with high FASN expression, which was indicated by high acetate uptake. To examine mechanisms, we established FASN knockdown prostate cancer cells by transduction of short-hairpin RNA against FASN and investigated the characteristics by analyses on morphology and cell behavior and microarray-based gene expression profiling. FASN inhibition not only suppressed cell proliferation but prevented pseudopodia formation and suppressed cell adhesion, migration, and invasion. FASN inhibition also suppressed genes involved in production of intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression. Collectively, our data demonstrated that uptake of radiolabeled acetate is a useful predictor of FASN-targeted therapy outcome. This suggests that [1-11C]acetate positron emission tomography (PET) could be a powerful tool to accomplish personalized FASN-targeted therapy by non-invasive visualization of tumor acetate uptake and selection of responsive tumors. FASN-targeted therapy could be an effective treatment to suppress multiple steps related to tumor progression in prostate cancers selected by [1-11C]acetate PET.

Monoclonal antibody RM2 as a potential ligand for a new immunotracer for prostate cancer imaging.

OBJECTIVES To investigate the potential of monoclonal antibody (mAb) RM2 as a ligand for a radioimmunotracer for prostate cancer imaging. METHODS Labeling was conducted with mAb RM2 and (125)I using the chloramine-T method. The cell study was conducted with PC-3 and LNCaP, which are prostate cancer cell lines, and MCF-7, which is a breast cancer cell line. The cells were treated or untreated with unlabeled mAb RM2 to block the haptoglobin-β chains expressed on the surface of the prostate cancer cells. (125)I-mAb RM2 was added into the cell culture media and cellular uptake of (125)I-mAb RM2 was evaluated at 1, 3 and 6 hours of incubation. For the in vivo biodistribution study, PC-3 cells were implanted in athymic male mice. The animals were injected intravenously with (125)I-mAb RM2. At 24, 48 and 72 hours after tracer injection, the animals were sacrificed and the activity levels of blood and tissue samples were determined. RESULTS The uptake of (125)I-mAb RM2 in the PC-3 and LNCaP cells increased according to the incubation time, while the uptake of (125)I-mAb RM2 in MCF-7 cells did not show any increase up to 6 hours. The increase of (125)I-RM2 uptake was not observed when the PC-3 and LNCaP cells were pre-treated with unlabeled RM2. In the biodistribution studies, (125)I-mAb RM2 showed marked uptake into the implanted PC-3 cells. In PC-3 tumor-bearing mice, the tumor muscle ratio of (125)I-RM2 was increased for up to 72 hours in a time-dependent manner. CONCLUSIONS (125)I-mAb RM2 showed excellent prostate cancer cell targeting in vitro and in vivo. Therefore, mAb RM2 seems to be a potential candidate for an immunoligand for prostate cancer imaging.

MP29-12 PRECLINICAL ASSESSMENT OF EARLY THERAPEUTIC EFFECT OF SUNITINIB ON RENAL CELL CARCINOMA USING 18F-FLUOROTHYMIDINE POSITRON EMISSION TOMOGRAPHY IN A XENOGRAFT MODEL

vaccines. Clinical and immunologic results vary. We have recently shown that genomic profiling of peripheral blood mononuclear cells (PBMC) may predict response. Transcription factors play a key role in the myelopoesis of monocytes and DC. We hypothesized that the profile of key transcription factors plays a key role in the efficacy of DC vaccines. METHODS: Monocytes from RCC patients and healthy donors were isolated via cold agglutination or ELUTRA. For some experiments, DC were matured with GM-CSF/ IL-4 and a variety of maturation protocols (TNF-alpha, cytokine cocktail, Flt-3).RNA was extracted using QIAGEN RNeasy Mini Kit. It’s concentration and quality was assessed using Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent Technologies). Only high quality RNA (RIN > 7.2) was used for reverse transcription (Bio-Rad iScript cDNA synthesis kit) and real time qPCR (in triplicates using ABI SYBR-Select Master Mix with target specific primers and the ABI 7900HT Thermocycler). The expression of key transcription factors IRF-4, IRF-8, IL-10, IL-12 were analyzed using previously reported primer pairs. To determine relative concentrations, cycle threshold (Ct) values were compared to the reference gene GAP-DH. The statistical analysis was conducted in JMP 11 from SAS. RESULTS: Healthy donors demonstrated a significant variation of expression levels of all transcription factors examined. When DC were matured with a variety of maturation protocols, these resulted in significant differences in expression of all transcription factors. The greatest variations were observed in the expression levels of IRF-4, IL10 and IL-12. The greatest induction of IRF-4 and IL-12 and lowest induction of IL-10 were observed with the cytokine cocktail. In an IFNgamma release assay, these cells also demonstrated the greatest T cell stimulatory capacity. RCC patient monocytes showed showed significantly lower expression of IRF-4 and IRF-8 than healthy age-adjusted donor monocytes (p1⁄40.004 and 0.0214, respectively). Immunotherapy resulted in upregulation of IL-10 when compared to pretreatment mRNA levels (p1⁄40.007). CONCLUSIONS: In summary, this is the first study to assess the profile of key transcription factors as predictors for DC vaccine quality. We demonstrate significant inter-individual variability in monocytes prior to DC vaccine and throughout the generation of DC vaccines. These data may lead to improved cellular anti-cancer vaccines.