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MP24-13 WITHDRAWN: RECURRENT PROSTATE CANCER GENOMIC ALTERATIONS PREDICT RESPONSE TO BRACHYTHERAPY TREATMENT
THE JOURNAL OF UROLOGYâ
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(miRNAs) in these processes has not been fully elucidated. miRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression and are predicted to regulate over 30% of human genes. Here we investigate the impact of 810 different miRNAs on PCa cell biology and sensitivity to radiation therapy. We anticipate that these miRNAs may be targets or therapeutics for enhancing the radiation therapy of PCa. METHODS: The Metridia Luciferase (MLuc) gene has been placed under the control of the human b-actin promoter and enhancer. MLuc activity of stably transfected PCa cell models, such as LNCaPMLuc, corresponded linearly with viable cell number. On day 0, 810 human miRNA mimics were transfected into LNCaP-MLuc cells. On day 2, one group of cells was irradiated with 4 Gy, while the other group remained untreated. On day 13, MLuc activity was examined to quantify cell viability and therapeutic effect, normalizing to the control miRNA. Toxic miRNAs, which increased cell death by over 50% in the absence of irradiation, were excluded from the analysis. Radiosensitizing or radioprotective miRNAs were defined as those which increased cell death by over 80% or increased cell survival by over 2 fold, respectively. Radiosensitizing miRNAs were validated in multiple PCa cell models. RESULTS: LNCaP-MLuc cells were applied to the miRNA viability and radiation sensitizing screen in duplicate, and the replicates were highly reproducible (R2¼0.75). Among 810 miRNAs, 127 were found to be toxic. 122 miRNAs were identified as radiosensitizing, and 75 were identified as radioprotective miRNAs. miRNAs belonging to the same families induced matched radiosensitization phenotypes. For example, miRNAs from the miR-15/16, miR-34/449 and miR-1/133 families were each observed to be radiation sensitizing, whereas miRNAs from the miR-106b family were found to be radioprotective. Over half of the top radiosensitizing miRNAs were confirmed to sensitize PC3-MLuc cells to radiation therapy. Additional miRNAs were validated by Clonogenic assay in DU145 cells. CONCLUSIONS: A series of high-throughput functional screening have identified miRNAs capable of regulating PCa cell radiosensitivity. These miRNAs may have therapeutic potential in the treatment of PCa with radiation therapy. Source of Funding: 5P50CA058236-15 (Nelson, WG), NIH/ NCI and The Patrick C Walsh Cancer Research Fund
MP24-13 WITHDRAWN RECURRENT PROSTATE CANCER GENOMIC ALTERATIONS PREDICT RESPONSE TO BRACHYTHERAPY TREATMENT Jacqueline Fontugne, Daniel Lee*, New York, NY; Chiara Cantaloni, Trento, Italy; Christopher Barbieri, New York, NY; Orazio Caffo, Trento, Italy; Esther Hanspeter, Guido Mazzoleni, Bolzano, Italy; Paolo Dalla Palma, Trento, Italy; Mark Rubin, New York, NY; Giovanni Fellin, Trento, Italy; Juan Miguel Mosquera, New York, NY; Mattia Barbareschi, Bolzano, Italy; Francesca Demichelis, Trento, Italy
MP24-14 METABOLIC SHIFT UNDER HYPOXIC ENVIRONMENT CAUSES RESISTANCE TO MTOR INHIBITOR IN HUMAN CASTRATION RESISTANT PROSTATE CANCER. Yota Yasumizu*, Takeo Kosaka, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Microenvironmental regulation of PI3K/Akt/mTOR signaling pathways and the efficacy of PI3K/mTOR inhibitors in hypoxic conditions have not been fully characterized yet. Recently we demonstrated that the activated PI3K/Akt/ mTOR signaling pathway induced by androgen deprivation therapy or docetaxel explains in part the aggressiveness of castration resistant prostate cancer (CRPC). The aim of this study was to explore the efficacy of a dual PI3K and mTOR inhibitor and mTOR inhibitor for docetaxel resistant CRPC under hypoxia. METHODS: Human CRPC cell lines: C4-2 and C4-2AT6 cell lines were used in this study. The C4-2AT6 cell line was established
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
from C4-2 cells grown in androgen ablated serum for six months. We examined the changes in PI3K/Akt/mTOR pathway and hypoxia-related proteins under hypoxic conditions by using the dual PI3K and mTOR inhibitor NVP-BEZ235, and the mTOR inhibitor everolimus. RESULTS: We investigated the expression of phosphorylated status in PI3K/Akt/mTOR pathway, HIF-1a, and pyruvate kinase isoform M2 (PKM2) in C4-2 and C4-2AT6 cells cultured under normoxia or hypoxia. PKM2 inactivates conversion of phosphoenolpyruvate to pyruvate and contributes to a metabolic shift that is required to cope with a hypoxic environment. Although under normoxia C4-2 and C4-2AT6 cells expressed elevated phosphorylated Akt (pAkt), phosphorylated S6 (pS6) and PKM2, a 5% hypoxic environment further induced expression of these proteins. These results indicate the PI3K/Akt/mTOR pathway is induced under hypoxic conditions. Next, we examined the cytotoxic effect of NVP-BEZ235 and everolimus under hypoxia by direct cell count. The relative cell number for treatment with 500nM NVP-BEZ235 and 100nM everolimus under 5% hypoxia compared to control was 0.46 0.04 and 0.91 0.11, respectively, while the relative cell number for treatment with the same doses of these drugs under normoxia was 0.47 0.05 and 0.68 0.08, respectively. NVP-BEZ235 showed a cytotoxic effect under hypoxia as well as normoxia (p<0.01). However, everolimus showed little cytotoxic effect under hypoxia. Western blot analysis revealed NVP-BEZ235 inhibited the expression of pAkt, pS6 and PKM2 in a dose-dependent manner. Everolimus did not inhibit the expression of pAkt or PKM2, although pS6 was fully inhibited. These results suggest that up-regulation of PKM2 is one possible mechanism of resistance to mTOR inhibitors in CRPC. CONCLUSIONS: A hypoxic microenvironment modulates the sensitivity of mTOR inhibitors through PKM2 expression. Source of Funding: none
MP24-15 THERMOTHERAPY WITH MAGNETIC CATIONIC LIPOSOMES POWERFULLY SUPPRESSES PROSTATE CANCER BONE METASTASIS IN A NOVEL RAT MODEL Daichi Kobayashi*, Komono tyou, Japan; Noriyasu Kawai, Shinya Sato, Toshiki Etani, Taku Naiki, Kenji Yamada, Yosuke Ikegami, Ryosuke Ando, Hiromichi Naruyama, Kazuhiro Kanemoto, Katsuhiro Fukuta, Daisuke Nagata, Hidetoshi Akita, Yoshihiro Hashimoto, Keiichi Tozawa, Nagoya City, Japan; Tohru Mogami, Komono tyou, Japan; Takehiko Okamura, Satoru Takahashi, Kenjiro Kohri, Nagoya City, Japan INTRODUCTION AND OBJECTIVES: Bone metastasis is a serious problem for prostate cancer patients, and the anticancer drug docetaxel (DTX) does not effectively treat this condition. Therefore, new therapies are urgently required. At the American Urological Association annual meetings in 2007, 2008, and 2010, we reported the therapeutic effects of our magnetite nanoparticles conjugated with cationic liposomes (MCLs). Here, we examined the therapeutic potential of MCLs for bone metastasis from prostate cancer compared to conventional DTX therapy in a novel rat model that allows tumor immunity evaluation. METHODS: Prostate tumor tissues were transplanted into the femurs of 4 groups of model rats: control, MCL, DTX, and MCL + DTX. Tumors were injected with MCLs, and alternating magnetic field (AMF) ^ MCLs (33 irradiation was performed thrice a week. We poured 200 f El mg/ml) into the tumor center just before each AMF exposure from 4 directions. DTX (10 mg/kg) was administered transcaudally. Tumor proliferation and bone destruction were evaluated by examining proliferating cell nuclear antigen positivity, computed tomography images, and CD68-positive cell numbers. Tumor immunity was evaluated by and heat assessing interleukin-2 (IL-2), interferon-gamma (IFN-f A), shock protein (HSP) 70 expression and determination of the CD8positive lymphocyte number. RESULTS: We successfully established a novel femur metastasis model of prostate cancer. This model allows long-term evaluation of bones and immune function. Tumor proliferation and bone destruction were significantly lower in the MCL and MCL + DTX groups than in
e264
(miRNAs) in these processes has not been fully elucidated. miRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression and are predicted to regulate over 30% of human genes. Here we investigate the impact of 810 different miRNAs on PCa cell biology and sensitivity to radiation therapy. We anticipate that these miRNAs may be targets or therapeutics for enhancing the radiation therapy of PCa. METHODS: The Metridia Luciferase (MLuc) gene has been placed under the control of the human b-actin promoter and enhancer. MLuc activity of stably transfected PCa cell models, such as LNCaPMLuc, corresponded linearly with viable cell number. On day 0, 810 human miRNA mimics were transfected into LNCaP-MLuc cells. On day 2, one group of cells was irradiated with 4 Gy, while the other group remained untreated. On day 13, MLuc activity was examined to quantify cell viability and therapeutic effect, normalizing to the control miRNA. Toxic miRNAs, which increased cell death by over 50% in the absence of irradiation, were excluded from the analysis. Radiosensitizing or radioprotective miRNAs were defined as those which increased cell death by over 80% or increased cell survival by over 2 fold, respectively. Radiosensitizing miRNAs were validated in multiple PCa cell models. RESULTS: LNCaP-MLuc cells were applied to the miRNA viability and radiation sensitizing screen in duplicate, and the replicates were highly reproducible (R2¼0.75). Among 810 miRNAs, 127 were found to be toxic. 122 miRNAs were identified as radiosensitizing, and 75 were identified as radioprotective miRNAs. miRNAs belonging to the same families induced matched radiosensitization phenotypes. For example, miRNAs from the miR-15/16, miR-34/449 and miR-1/133 families were each observed to be radiation sensitizing, whereas miRNAs from the miR-106b family were found to be radioprotective. Over half of the top radiosensitizing miRNAs were confirmed to sensitize PC3-MLuc cells to radiation therapy. Additional miRNAs were validated by Clonogenic assay in DU145 cells. CONCLUSIONS: A series of high-throughput functional screening have identified miRNAs capable of regulating PCa cell radiosensitivity. These miRNAs may have therapeutic potential in the treatment of PCa with radiation therapy. Source of Funding: 5P50CA058236-15 (Nelson, WG), NIH/ NCI and The Patrick C Walsh Cancer Research Fund
MP24-13 WITHDRAWN RECURRENT PROSTATE CANCER GENOMIC ALTERATIONS PREDICT RESPONSE TO BRACHYTHERAPY TREATMENT Jacqueline Fontugne, Daniel Lee*, New York, NY; Chiara Cantaloni, Trento, Italy; Christopher Barbieri, New York, NY; Orazio Caffo, Trento, Italy; Esther Hanspeter, Guido Mazzoleni, Bolzano, Italy; Paolo Dalla Palma, Trento, Italy; Mark Rubin, New York, NY; Giovanni Fellin, Trento, Italy; Juan Miguel Mosquera, New York, NY; Mattia Barbareschi, Bolzano, Italy; Francesca Demichelis, Trento, Italy
MP24-14 METABOLIC SHIFT UNDER HYPOXIC ENVIRONMENT CAUSES RESISTANCE TO MTOR INHIBITOR IN HUMAN CASTRATION RESISTANT PROSTATE CANCER. Yota Yasumizu*, Takeo Kosaka, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Microenvironmental regulation of PI3K/Akt/mTOR signaling pathways and the efficacy of PI3K/mTOR inhibitors in hypoxic conditions have not been fully characterized yet. Recently we demonstrated that the activated PI3K/Akt/ mTOR signaling pathway induced by androgen deprivation therapy or docetaxel explains in part the aggressiveness of castration resistant prostate cancer (CRPC). The aim of this study was to explore the efficacy of a dual PI3K and mTOR inhibitor and mTOR inhibitor for docetaxel resistant CRPC under hypoxia. METHODS: Human CRPC cell lines: C4-2 and C4-2AT6 cell lines were used in this study. The C4-2AT6 cell line was established
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
from C4-2 cells grown in androgen ablated serum for six months. We examined the changes in PI3K/Akt/mTOR pathway and hypoxia-related proteins under hypoxic conditions by using the dual PI3K and mTOR inhibitor NVP-BEZ235, and the mTOR inhibitor everolimus. RESULTS: We investigated the expression of phosphorylated status in PI3K/Akt/mTOR pathway, HIF-1a, and pyruvate kinase isoform M2 (PKM2) in C4-2 and C4-2AT6 cells cultured under normoxia or hypoxia. PKM2 inactivates conversion of phosphoenolpyruvate to pyruvate and contributes to a metabolic shift that is required to cope with a hypoxic environment. Although under normoxia C4-2 and C4-2AT6 cells expressed elevated phosphorylated Akt (pAkt), phosphorylated S6 (pS6) and PKM2, a 5% hypoxic environment further induced expression of these proteins. These results indicate the PI3K/Akt/mTOR pathway is induced under hypoxic conditions. Next, we examined the cytotoxic effect of NVP-BEZ235 and everolimus under hypoxia by direct cell count. The relative cell number for treatment with 500nM NVP-BEZ235 and 100nM everolimus under 5% hypoxia compared to control was 0.46 0.04 and 0.91 0.11, respectively, while the relative cell number for treatment with the same doses of these drugs under normoxia was 0.47 0.05 and 0.68 0.08, respectively. NVP-BEZ235 showed a cytotoxic effect under hypoxia as well as normoxia (p<0.01). However, everolimus showed little cytotoxic effect under hypoxia. Western blot analysis revealed NVP-BEZ235 inhibited the expression of pAkt, pS6 and PKM2 in a dose-dependent manner. Everolimus did not inhibit the expression of pAkt or PKM2, although pS6 was fully inhibited. These results suggest that up-regulation of PKM2 is one possible mechanism of resistance to mTOR inhibitors in CRPC. CONCLUSIONS: A hypoxic microenvironment modulates the sensitivity of mTOR inhibitors through PKM2 expression. Source of Funding: none
MP24-15 THERMOTHERAPY WITH MAGNETIC CATIONIC LIPOSOMES POWERFULLY SUPPRESSES PROSTATE CANCER BONE METASTASIS IN A NOVEL RAT MODEL Daichi Kobayashi*, Komono tyou, Japan; Noriyasu Kawai, Shinya Sato, Toshiki Etani, Taku Naiki, Kenji Yamada, Yosuke Ikegami, Ryosuke Ando, Hiromichi Naruyama, Kazuhiro Kanemoto, Katsuhiro Fukuta, Daisuke Nagata, Hidetoshi Akita, Yoshihiro Hashimoto, Keiichi Tozawa, Nagoya City, Japan; Tohru Mogami, Komono tyou, Japan; Takehiko Okamura, Satoru Takahashi, Kenjiro Kohri, Nagoya City, Japan INTRODUCTION AND OBJECTIVES: Bone metastasis is a serious problem for prostate cancer patients, and the anticancer drug docetaxel (DTX) does not effectively treat this condition. Therefore, new therapies are urgently required. At the American Urological Association annual meetings in 2007, 2008, and 2010, we reported the therapeutic effects of our magnetite nanoparticles conjugated with cationic liposomes (MCLs). Here, we examined the therapeutic potential of MCLs for bone metastasis from prostate cancer compared to conventional DTX therapy in a novel rat model that allows tumor immunity evaluation. METHODS: Prostate tumor tissues were transplanted into the femurs of 4 groups of model rats: control, MCL, DTX, and MCL + DTX. Tumors were injected with MCLs, and alternating magnetic field (AMF) ^ MCLs (33 irradiation was performed thrice a week. We poured 200 f El mg/ml) into the tumor center just before each AMF exposure from 4 directions. DTX (10 mg/kg) was administered transcaudally. Tumor proliferation and bone destruction were evaluated by examining proliferating cell nuclear antigen positivity, computed tomography images, and CD68-positive cell numbers. Tumor immunity was evaluated by and heat assessing interleukin-2 (IL-2), interferon-gamma (IFN-f A), shock protein (HSP) 70 expression and determination of the CD8positive lymphocyte number. RESULTS: We successfully established a novel femur metastasis model of prostate cancer. This model allows long-term evaluation of bones and immune function. Tumor proliferation and bone destruction were significantly lower in the MCL and MCL + DTX groups than in