Male Nude Mice Research Articles

Abstract 380: Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer.

Abstract Angiogenesis is required for all tumors to grow and metastasize and is mainly induced by the vascular endothelial growth factor (VEGF). Alternative spliced isoforms associated with cancer progression have been described in all areas of hallmarks of cancer, including angiogenesis. VEGF mRNA is alternatively spliced at the terminal exon, to produce two families of isoforms. The pro- angiogenic family, VEGFxxx, was shown to be up-regulated in all cancers studied so far, where as the anti- angiogenic family is preferentially expressed in normal non-angiogenic tissues, but down-regulated in most cancers. One of the factors involved in the regulation of VEGF alternative splicing is SRPK1. This is a protein kinase that phosphorylates the splicing factor SRSF1 and favours the production of the pro-angiogenic VEGF isoform. We therefore tested the hypothesis that down - regulation of SRPK1 in prostate cancer cells would increase the production of the anti-angiogenic isoform, preventing angiogenesis and tumor growth. PC-3, DU145 and LnCap prostate cancer cells showed an increased expression of SRPK1 when compared to Primary Prostate epithelial cells. Stable SRPK1 knock-down (KD) in PC-3 and LnCap prostate cancer cells was obtained using a lentiviral system (Dharmacon). This resulted in a reduction in splice factor expression, mainly SRSF1, and induced a switch in the expression of VEGF towards the anti-angiogenic isoform, VEGF165b, when compared to control cells (transfected with non-targeting shRNA). PC-3 SRPK1 KD and control cell lines were injected subcutaneously in male nude mice. Knock down of SRPK1 resulted in an inhibition of tumour growth, as shown by the significant difference in tumor size between the SRPK1 KD (250.0 ± 82.28 n=9) and control cell line (994.7 ± 243.5 n=9)(Day 29 post-injection). In addition to the reduction in tumor volume, SRPK1 inhibition resulted in decrease in vascular density of the tumors, as shown by the significant reduction in blood vessel number in PC-3 SRPK1 KD (2.714 ± 0.3686 n=7) mice compared with control (4.292 ± 0.5426 n=8). These in vitro and in vivo data suggest that the regulation of VEGF splicing by SRPK1 alters prostate cancer tumour growth and angiogenesis. Citation Format: Athina Mavrou, Sebastian Oltean, Dave Bates. Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2013-380

Abstract 2813: EP4 antagonist suppresses bone metastasis in prostate cancer.

Abstract Introduction Bone metastasis is one of the most common complications in clinical intractable castration-resistant prostate cancer (CRPC) and advanced prostate cancer period. The invasion ability of cancer cells was thought to have important relationship with the potential of cancer metastasis. EP4 receptor is overexpressed in several cancers, and enhanced EP4 receptor signaling has been previously shown to correlate with the invasion of several different cancer types. In present study, we found that EP4 antagonist can in vitro suppress the invasion phenotype abilities of CRPC PC3 cells, LNCaP cells with EP4 overexpressed which have the tendency to castration-resistance and in vivo suppress the CRPC bone metastasis animal models. Materials and methods Materials In vitro: PC3 cell and LNCaP cell in vivo: PC3/Luc cell. EP4-specific antagonist: ONO-AE3-208 Methods RNA interference was used to stably transfected EP4 to LNCaP with the pcDNA3.1-EP4, so as to be the LNCaP with vector control (LNCaP-mock) and LNCaP with EP4 overexpressed (LNCaP/EP4+) as described in our previous article. Effect on cell proliferation was determined firstly by the MTT assay. Transwell invasion assay was used to in vitro examine the metastasis abilities of PC3 cells, LNCaP cells, LNCaP/EP4+ and all after EP4 antagonist affected. Cells with ONO-AE3-208 at the final concentration of 0.1μmol/L, 1μmol/L and 10μmol/L were administrated to be the experimental group. To observe the effect of EP4 antagonist on the bone metastasis of prostate cancer in vivo, PC3/Luc cells were inoculated into the left cardiac ventricle of 5-week-old male nude mice. The mice developed osteolytic lesions with a mean endpoint at 52±7 days.To assess whether EP4 antagonist suppresses bone metastasis, ONO-AE3-208 (10 mg/kg/d) was given to the animals 5 days a week I.p. which the same volume of DDW was given as the control.To detect PC3 cell dissemination, bioluminescent imaging (BLI) was applied using a cooled CCD camera. We evaluated the efficacy of ONO-AE3-208 by measuring the photon counts of the metastatic lesions in the mandible and both hip joints in a blinded manner. Results The invasion assay showed that ONO-AE3-208 can inhibit the invasive abilities of both PC3 and LNCaP/EP4+ in a dose-dependent manner (p < 0.01). In animal experiment, metastatic bone lesions in the control group progressed while the photon emission was significantly suppressed in the ONO-AE3-208 treatment group during the observation period from day 28 every 7 days (p < 0.01). Conclusion We demonstrated that EP4 antagonist can in vitro inhibit the invasive abilities of CRPC PC3 and LNCaP/EP4+ cells. The administration of it can also suppress the bone metastasis of CRPC in vivo. Citation Format: Song Xu, Takayuki Goto, Takeshi Yoshikawa, Zhengyu Zhang, Liming Wang, Naoki Terada, Osamu Ogawa. EP4 antagonist suppresses bone metastasis in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2813. doi:10.1158/1538-7445.AM2013-2813

Abstract 1380: In vivo evidence for prior manipulation therapy-induced metastasis of primary osteosarcoma.

Abstract Our previous clinical study demonstrated that manipulation therapy (MT) on osteosarcoma (OS) patients prior to diagnosis resulted in poor prognosis after surgical treatment. This study was aimed to provide in vivo evidence of MT-induced metastasis in primary osterosarcoma. Eight-week-old male GFP-labeled human OS cells-transferred nude mice were randomly allocated into 2 groups, namely, MT (+) and MT (-) groups. MT was conducted with repeated massage on tumor site twice a week for 7 or 15 weeks. The parameters evaluated were x-ray diagnosis, micro-PET/CT scan, histopathology and serum metalloproteinase 9 (MMP9) level. The results showed that MT (+) mice showed a decreased body weight (30.5±0.65g) and an increased tumor volume (8.3±1.18 mm3) compared to MT (-) group with body weight (35.8±0.40g, p<0.0001) and tumor volume (3.9±1.34mm3, p=0.038), respectively. There was an increased signal intensity over lymph node region of hind limb by micro-PET/CT and the GFP-labeled human OS cells were detected in the lung and bilateral lymph nodes in MT (+) group, while there were no such findings in MT (-) group. The serum MMP9 level was higher in MT (+) group (27.1±1.29 ng/ml) than in MT (-) group (17.8±1.97 ng/ml, p=0.048). Taken previous clinical observation and the present in vivo evidence together, we conclude that physicians should pay more attention on those patients who seek MT before diagnosis or during treatment for osteosarcoma. Citation Format: Jir-You Wang, Po-Kuei Wu, Chih-Hsueh Chen, Chuen-Chuan Yen, Giun-Yi Hung, Shih-Chieh Hung, Wei-Ming Chen, Chien-Lin Liu, Tain-Hsiung Chen. In vivo evidence for prior manipulation therapy-induced metastasis of primary osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1380. doi:10.1158/1538-7445.AM2013-1380

Abstract 3697: Molecular link that associates high fat diet and prostate tumor growth.

Abstract Prostate Cancer (PCa) is one of the most common invasive tumors in men. Epidemiological studies indicate that diet and overweight are important factors implicated in prostate carcinogenesis. Obesity is associated with PCa aggressiveness, poorer prognosis and increased mortality. Breast cancer susceptibility gene 1 (BRCA1) interacts with several transcriptional regulators to modulate the androgen receptor (AR) signaling in PCa cell lines. Germline mutations in this gene increase breast cancer risk and are associated with high grade PCa. Previously, it had been reported that C-terminal Binding Protein 1 (CtBP1) acts as a switch to control BRCA1 transcription in response to the metabolic status of the cells. The release of CtBP1 from BRCA1 promoter through estrogen induction and high NAD+/NADH ratio (similar to high caloric intake) increases BRCA1 transcription in breast cancer cells. The aim of this work was to assess the effect of androgens and/or high fat diet over the BRCA1/CtBP1 axis and PCa tumor growth. We found that BRCA1 and CtBP1 proteins associate to BRCA1 proximal promoter region in PC3 cells and suppress BRCA1 transcription. Testosterone stimulation released these factors from BRCA1 promoter increasing its transcription. To assess whether this activation is mediated by testosterone or the estrogen, synthesized from testosterone by the aromatase (CYP19A1), we investigated this mechanism in the presence of letrozol (LTZ), an aromatase inhibitor. We found that LTZ abolished BRCA1 induction by testosterone, suggesting that BRCA1 activation is mediated by estrogen in these cells. Furthermore, we generated PC3 cell lines transfected with pcDNA3-CtBP1 (PC3-CtBP1) or shRNA-CtBP1 (PC3-shCtBP1) plasmids, to overexpress or knock down CtBP1 expression, respectively. CtBP1 induction decreased BRCA1 expression in these cells and this effect was reverted by CtBP1 depletion. In addition, PC3-CtBP1 cells showed increased clonogenic capacity and proliferation compared to PC3-shCtBP1 cells. Moreover, we developed an in vivo model to investigate the effect of high caloric diet on PCa growth after CtBP1 modulated-expression. High fat or control diet fed male nude mice were inoculated with PC3-CtBP1 and PC3-shCtBP1 stable cells. We found that CtBP1 depleted cells growing as xenografts in high fat diet fed mice dramatically decreased prostate tumor growth. Molecular analysis of tumors by RT-qPCR showed that CtBP1 depletion correlated with high BRCA1 expression. In addition, serum from high fat fed mice significantly induced PC3-CtBP1 cell proliferation in vitro. These results strongly suggest that the potential oncogenic role of CtBP1 is dependent on the caloric diet intake. Hence, BRCA1 regulation by CtBP1 provides an important molecular link between caloric intake and tumor suppressor expression. Citation Format: Cristian P. Moiola, Paola De Luca, Florencia Zalazar, Javier Cotignola, Estefania Labanca, Roberto Meiss, Elba S. Vazquez, Kevin Gardner, Adriana De Siervi. Molecular link that associates high fat diet and prostate tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2013-3697

Abstract 3946: Saracatinib inhibits human prostate cancer cell bone metastases in a xenograft model.

Abstract Background: In advanced prostate cancer (PCa), cells escape from the primary tumor and enter the bloodstream. These circulating cells preferentially target the axial skeleton and can form bone metastases. This results in weakened bones, spinal compressions, and fractures. Patients experience pain and other bone-related events that increase mortality and decrease quality of life. Therefore, identifying factors that lead to cancer progression, such as areas of hypoxia in the tumor microenvironment, will be crucial to developing strategies to inhibit the spread of malignant cells to distant sites. Efforts to interfere with early steps in the metastatic cascade have led to the development of promising small molecule targeting agents. One such agent is the tyrosine kinase inhibitor (TKI), saracatinib, shown to inhibit activation of Src and other kinases thought to be involved in cancer cell dissemination. The aim of these studies was to use aggressive, human PCa cells (PC-3ML), selected for their ability to form bone tumors in mice, to investigate the effects of various agents shown to interfere with metastatic activity. Methods: PC-3ML cells (a gift from A. Fatatis, Drexel University), expressing Green Fluorescent Protein (GFP) and luciferase (Luc), were injected into the left ventricle of male nude mice, using a closed-chest technique, to ensure that they bypass the lungs and circulate throughout the body. Prior to injection, cells were subjected to either hypoxia for 6 or pretreated for 24 hr with saracatinib. Immediately after injection, mice were placed in an in vivo imaging system (Xenogen IVIS, Caliper Life Sciences) to verify that the cells had disseminated throughout the body. This ensured that only successfully injected mice were randomization to treatment groups. Additional images were collected weekly thereafter to monitor the location and development of tumor nodules. Three weeks post-injection, mice were euthanized and tissues expressing Luc were collected, including bones of the legs, mandible and spine. The bones were fixed and decalcified, followed by flash freezing in OCT freezing medium. Serial frozen sections were then evaluated for GFP expressing cells using a fluorescent stereoscope. Results: Tumor cell deposits were almost always found in bone, frequently in the tibial-femoral joint, scapula-humerus joint, spine, and mandible. Mice injected with PC-3ML cells pretreated with saracatinib had fewer metastases and cells exposed to hypoxia for 6 hr prior to injection had more metastases, on average, than control cells, as predicted by in vitro assays. Conclusions: Intracardiac injection of GFP- and Luc- labeled PC-3ML cells resulted in skeletal metastases that could be monitored in mice over time using an in vivo imaging system and in tissue sections by fluorescent microscopy. The studies verified in vitro data showing that hypoxia can enhance, and saracatinib inhibit, metastatic activity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Saracatinib inhibits human prostate cancer cell bone metastases in a xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3946. doi:10.1158/1538-7445.AM2013-3946

Abstract 3919: A preclinical model of laparoscopy demonstrating the feasibility of detecting PSMA positive cells with a NIR fluorophore (YC-27): implications for strategies to decrease positive margins during prostatectomy.

Abstract Introduction: Depending on the stage there is approximately a 4-54% positive surgical margin (+SM) following prostatectomy. Patients with +SM face the associated expense and impaired functional outcomes of salvage radiotherapy. To circumvent this, a low-molecular weight urea-based near infrared (NIR) fluorophore (YC-27) was developed to target the prostate specific membrane antigen (PSMA) for use in intraoperative imaging. PSMA is a transmembrane protein expressed in normal prostate and upregulated in cancer. PSMA expression correlates with Gleason score, advanced stage and PSA. We optimized dosing and timing in a small animal model for laparoscopic resection of PSMA positive tumors. Secondly, we demonstrate the feasibility of applying such a system in a porcine model. Methods: PSMA positive and negative tumors were established contralaterally on the flank of athymic male nude mice. YC-27 was administered intravenously at varying doses (9.5, 19.1, 39.7 [ug/Kg]) and sequentially imaged using a custom built laparoscopic system to obtain probe kinetics (1, 2, 3, 4, 6, 8, 10, 24 hrs post IV injection). Arbitrary pixel count was used to evaluate dosing and timing for signal to noise ratio. NIR guided laparoscopic resection was performed on mice with established PSMA positive and negative tumors 6 hrs post injection of 39.7 ug/Kg YC-27. In a porcine model, prestained human xenografts generated in a murine host were implanted behind the peritoneum of the abdominal cavity, and exploratory NIR laparoscopy was performed. In a porcine model, the kidney was observed with our laparoscopic NIR system after intravenous YC-27 administration. Results: In a murine model, the highest signal to noise ratio was determined to occur 6 hrs after injecting 39.7 ug/Kg YC-27. Using these parameters, NIR guided laparoscopic surgery facilitated full resection of the xenograft. In a porcine model, exploratory laparoscopy with our NIR system allowed detection of a prestained tumor implanted behind the peritoneum. In another experiment, strong fluorescent signal was observed from the porcine kidney immediately after IV injection of YC-27 which may be attributed to specific binding to PSMA, renal clearance, or a combination of both. Conclusions: YC-27 allows intraoperative localization of PSMA in real time using a prototype NIR guided laparoscopic surgical system. Pilot studies in large animals demonstrated excellent potential for clinical translation. Citation Format: Brian P. Neuman, John B. Eifler, Mark Castanares, Wasim H. Chowdhury, Ying Chen, Martin G. Pomper, Ronald Rodriguez. A preclinical model of laparoscopy demonstrating the feasibility of detecting PSMA positive cells with a NIR fluorophore (YC-27): implications for strategies to decrease positive margins during prostatectomy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2013-3919

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

BackgroundColorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).Materials and MethodsHCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.ResultsBoth HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.ConclusionsWe demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.

Genistein reduced colon cancer metastasis to liver through regulating the expression of metastasis‐related genes in xenographed mice model

This study aimed at investigating the effect of genistein (Gen) on liver metastases derived from colon cancer. Male athymic nude mice were fed with western diet (W; calories%, fat 39%, carb 50%), W diet supplemented with 100ppm Gen (GL) or 500ppm Gen (GH). After 16 weeks, all the animals were intrasplenically injected with metastatic human colon cancer cells SW620 and sacrificed 8 weeks later. GL and GH mice showed lower liver metastases (36% and 17%) compared to W group (55% of mice with metastases). Mean size of metastases in the W group (4.7 mm2) was larger than that in GL (2.1 mm2) and GH (2.0 mm2) groups. To understand the molecular mechanism of the anti‐metastatic function of Gen, the expression of multiple metastasis‐related genes were determined by real‐time PCR. mRNA of NDRG1 gene in livers was greatly decreased by 50% and 60% in GL (p=0.005) and GH group (p=0.001) compared to W group. As NDRG1 has been reported as a biomarker for metastasis and poor prognosis in hepatocellular carcinoma, the down‐regulation of NDRG1 by Gen supplementation was corresponding to decreased liver metastasis. In summary, our study indicated that dietary supplementation of Gen suppressed colon cancer metastasis, which is associated with altered expression of metastasis‐related genes by the treatment.Grant Funding Source: university of illinois

Toxicological and Cancer‐Chemopreventive Evaluation of a Novel Soluble Silicate

Silicates are ubiquitous in nature and exhibit diverse speciation and unique physiochemical properties. Though abundant in diet, their biological role in animals is not clearly understood. In our previous study, we showed that a unique soluble species of silicate [Na8.2Si4.4H9.7O17.6] modulated stress response signaling pathways in vivo and may have implications in human health. Here we present results from toxicological and cancer chemopreventive evaluation of Na8.2Si4.4H9.7O17.6. Results suggest LD50 >;5000 mg/kg for acute oral toxicity of silicates in Sprague‐Dawley rats. Undiluted silicate was found to be non‐corrosive in rabbit skin corrosion study. A statistically significant reduction in fibroblast, platelet‐derived and vascular endothelial growth factor induced angiogenesis was observed in the chick embryo chorioallantoic membrane model at >;18.8 nM. A significant reduction in the size of the NM2C5 tumors was observed in vitro in the three‐dimensional tumor microenvironment model at a dosage of >;18.8 nM. At a dosage of >; 7.5 mM the silicate also decreased the tumor development and metastasis of LOX‐GFP human melanoma in Male NCr nude mice by ~15%. Our results suggest that soluble silicates may have potential cancer‐chemopreventive effects and merits further detailed investigation to understand mechanism of action.

982 COMBINED TARGETING OF PI3K/AKT AND AR PATHWAY WITH AZD5363 AND ENZALUTAMIDE INDUCES ANTICANCER ACTIVITY IN PRECLINICAL MODELS OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013982 COMBINED TARGETING OF PI3K/AKT AND AR PATHWAY WITH AZD5363 AND ENZALUTAMIDE INDUCES ANTICANCER ACTIVITY IN PRECLINICAL MODELS OF PROSTATE CANCER Paul Toren, Soojin Kim, Martin Gleave, and Amina Zoubeidi Paul TorenPaul Toren Vancouver, Canada More articles by this author , Soojin KimSoojin Kim Vancouver, Canada More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Amina ZoubeidiAmina Zoubeidi Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.564AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent advances in castrate resistant prostate cancer (CRPC) treatment using androgen receptor (AR) targeted therapies such as enzalutamide (MDV3100) have produced striking survival advantages in clinical studies. However, the next major clinical challenge that we will face is how to prevent and treat the emergence of MDV3100 resistant tumors that rapidly occur in some CRPC patients. We and others found that MDV3100 induces phospho-activation of AKT (pAKT) and that inhibition of AKT results in feedback signaling leading to activation of AR suggesting that inhibiting AKT alone is not a good strategy to combat MDV3100 resistance. Here we investigate the effect of dual targeting Akt using AZD5363 and the AR pathway using MDV3100. METHODS Human prostate cancer cell lines LNCaP, C4-2, 22RV1 and MDV3100 resistant cell line (MR49C) were treated with AZ5363, MDV3100 or AZ5363 plus MDV3100. Cell proliferation was assessed by crystal violet, cell cycle population was assessed by FACS analysis, and cell apoptosis was assessed by PARP cleavage and caspase-3 activity. AR transcriptional activity was assessed by probasin luciferase and AR signaling pathway was assessed by western blot and qRT-PCR. For in vivo anti-cancer activity, male athymic nude mice were injected s.c. with LNCaP cells and castrated after start of tumor growth. Animals were randomly selected for vehicle, AZD5363, MDV3100 and AZD5363+MDV3100 once CRPC was identified. Tumor volume and serum PSA are being monitored weekly. RESULTS We found that combination therapy was more potent than mono-therapy. AZD5363 and MDV3100 synergize and induce cell apoptosis showing PARP cleavage and caspase 3 activity as well as increase of subG1/G0 population in all cell lines including the MDV3100-resistant MR49C. Moreover, we found that MDV3100 abrogates AZD5363 induced up-regulation of AR transcriptional activity and up-regulation of AR dependent genes both at mRNA and protein levels. The effect of the combination in an in vivo model of prostate cancer progression is currently under evaluation. CONCLUSIONS Combined blockade of the AKT and AR pathway with AZD5363 and MDV3100 demonstrates significant induction of apoptosis and proliferation and provides a preclinical proof of principle for using these two drugs in the clinic in CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e403 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Paul Toren Vancouver, Canada More articles by this author Soojin Kim Vancouver, Canada More articles by this author Martin Gleave Vancouver, Canada More articles by this author Amina Zoubeidi Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

973 A NEW STRATEGY FOR CASTRATION RESISTANT PROSTATE CANCER WITH TARGETING TO FEEDBACK MECHANISM OF PI3K/AKT/MTOR PATHWAY

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013973 A NEW STRATEGY FOR CASTRATION RESISTANT PROSTATE CANCER WITH TARGETING TO FEEDBACK MECHANISM OF PI3K/AKT/MTOR PATHWAY Yota Yasumizu, Akira Miyajima, Takeo Kosaka, Yasumasa Miyazaki, Suguru Shirotake, Eiji Kikuchi, and Mototsugu Oya Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki Tokyo, Japan More articles by this author , Suguru ShirotakeSuguru Shirotake Saitama, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.555AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The clinical trials of mTORC1 inhibiton in CRPC have failed to demonstarate anti-tumor effect despite the deficit of PTEN observed in about 70% of castration resistant prostate cancer (CRPC) with metastasis. The up-regulation of phosphorylated Akt (pAkt) through mTORC2 is reported as one factor of mTORC1 inhibitor resistance. In this study we aimed to establish a new strategy for docetaxel resistant CRPC, with a focus on regulatory system in PI3K/Akt/mTOR signaling pathway. METHODS The efficacy of PI3K/Akt/mTOR signaling pathway inhibition by the mTORC1 inhibitor: RAD001 and the PI3K and mTORC1/2 dual inhibitor: NVP-BEZ235 was examined in C4-2AT6 cells in vivo and in vitro. C4-2AT6 cells are a new CRPC cell line established by our laboratory under androgen ablation conditions and shows docetaxel resistance. RESULTS To examine the inhibitory effect of NVP-BEZ235 or RAD001, we performed the western blotting using C4-2AT6 cells. The expression of pAkt and phosphorylated S6 ribosomal protein (pS6) were significantly inhibited by 50nM NVP-BEZ235. On the other hand, the expression of pAKT was not inhibited by the treatment with RAD001, although the expression of pS6 was inhibited by 10nM RAD001. Next we tested the effect of NVP-BEZ235 or RAD001 to C4-2AT6 cells by WST assay. The mean cell viability at 24 h of treatment with 50nM NVP-BEZ235 and 10nM RAD001 was 59.2&undefined;} 6.4% and 61.2&undefined;} 7.2%, respectively. There was no significant difference between the two cell viability. The cell viability with 50nM NVP-BEZ235 and 5nM docetaxel was 41.2&undefined;} 5.2% and the cell viability with 10nM RAD001 and 5nM docetaxel was 50.1&undefined;} 4.8%. The combination of NVP-BEZ235 and docetaxel showed significantly higher anti-tumor effect than the combination of RAD001 and docetaxel (p<0.05). In vivo, we assigned castrated male nude mice to 4 groups; control, NVP-BEZ235 (12.5mg/kg/day), docetaxel (2mg/kg), and NVP-BEZ235 combined with docetaxel. We examined the time course changes in C4-2AT6 tumor growth with NVP-BEZ235 and docetaxel. The tumor volume of NVP-BEZ235 at day15 was 89.8&undefined;} 9.8%, and the tumor volume of docetaxel was 81.2&undefined;} 6.4%, compared to the control group. While the tumor volume of combined treatment was 6.1&undefined;} 5.5% of control. The combination therapy exhibited marked suppression of tumor growth compared to control, NVP-BEZ235 monotherapy, or docetaxel monotherapy (p<0.05). CONCLUSIONS The combination of NVP-BEZ235 and docetaxel showed significant anti-tumor effects in CRPC, suggesting the efficacy of PI3K/mTORC1/2 dual inhibitor for regulation of PI3K/Akt/mTOR pathway in CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e400 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yota Yasumizu Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Yasumasa Miyazaki Tokyo, Japan More articles by this author Suguru Shirotake Saitama, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

321 ANTISENSE INHIBITOR TARGETING FULL LENGTH ANDROGEN RECEPTOR AND ITS SPLICE VARIANTS SUPPRESSES MDV3100 RESISTANT PROSTATE CANCER CELL GROWTH

in different cancer types. We previously found that miR-125b directly targets p53, Bak1 and Puma, and promoted prostatic tumor growth in both intact and castrated mice. In this study, we further underline the clinical importance of miR-125b by detecting miR-125b levels in prostate cancer (CaP) specimens. In addition, we conducted animal experiments to determine whether miR-125b has potential as a therapeutic target for treatment of CaP. METHODS: For detection of miR-125b expression, total RNA was isolated from cancer cell-enriched, fresh-frozen CaP tissues, as well as fresh-frozen BPH specimens, using TRIzol reagent. The miR125b level was measured using qPCR. Animal experiments were performed according to the protocols approved by the Animal Care and Use Committee at UCD, CA USA. Male nude mice implanted with testosterone pellets were inoculated s.c with CWR22 CaP cell suspensions and then treated with polyethylenimines (PEI)-complexed antimiR-125b (PEI-anti-miR-125b) or PEI-anti-miR-125b plus Taxol. PEI can form complexes with miRNA, increase internalization and facilitate miRNA release into cytoplasm. RESULTS: 133 prostate specimens (46 BPHs and 87 CaPs) were analyzed for their expression of miR-125b. The average miR125b level was 3.0-fold greater in CaPs than in BPHs. In 42 CaPs having Gleason score available, the miR-125 levels correlate with Gleason scores (103 18 relative fold in Gleason score 5-6 vs 160 16 in Gleason score 7-10, p 0.03), suggesting that miR-125b contributes to pathological progression of CaP. In animal studies, mice were treated with PEI-anti-miR-125b (10 g/day, thrice a week for 4 weeks) or PEI-anti-miRNA control, and significant tumor-inhibitory effects was observed in anti-miR-125b treatment group compared to control group. In addition, mice bearing CWR22 tumors were treated with PEI-antimiR-125b plus Paclitaxel (20 mg/kg/week for 4 weeks). It was found that anti-miR-125b significantly increased the therapeutic efficiency of Taxol. CONCLUSIONS: Data from qPCR provide evidence that dysregulation of miR-125b is common in CaP patients and the levels of miR-125b are correlated with pathological progression of CaP. Additionally, results from animal studies show that anti-miR-125b inhibited growth of CWR22 tumors and sensitized CaP cells to Taxol, suggesting that miR-125b can be used as a target for improved CaP treatment.

Effect of thalidomide on the proliferation of hepatoma cells assessed by osteopontin levels in nude mice

The aim of the present study was to investigate the inhibitory effects of thalidomide in the hepatocellular carcinoma nude mouse model in order to provide new insights into a comprehensive clinical intervention for hepatocellular carcinoma. MHCC97 cells were routinely cultured, passaged and adjusted to a single cell suspension with a concentration of 2×107/ml. Six-week-old, BALB/C male nude mice were anesthetized and fixed in the prone position, then a subcapsular injection of the single cell suspension was administered into the spleen and their abdomens were closed. A laparotomy and left hepatic lobectomy was performed 14 days later and the abdomens were closed once again. Subsequent to the establishment of the hepatocellular carcinoma model, the nude mice were randomly divided into three groups, each consisting of 12 mice. The early intervention group were immediately provided with the post-operative thalidomide intervention, the late intervention group were provided with the post-operative thalidomide intervention one week subsequent to the surgery, and the negative control group were provided with a placebo intervention (0.9% physiological saline). Each intervention was continuously administered once per day for one week. The osteopontin (OPN) content of the liver tumors was detected using immunohistochemistry. The data were analyzed using an analysis of variance (ANOVA) test. There were significant differences in the OPN levels of the tumors among the early intervention, late intervention and negative control groups. Thalidomide may inhibit the generation of OPN and thereby inhibit the infiltration and metastasis of tumors; the immediate use of thalidomide following hepatectomy in the present study may block the invasion and metasis for liver cancer more effectively.

Synthesis and Evaluation of a Targeted Nanoglobular Dual-Modal Imaging Agent for MR Imaging and Image-Guided Surgery of Prostate Cancer

To synthesize and evaluate a peptide targeted nanoglobular dual modal imaging agent specific to a cancer biomarker in tumor stroma for MRI and fluorescence visualization of prostate tumor in image-guided surgery. A peptide (CGLIIQKNEC, CLT1) targeted generation 2 nanoglobular (polylysine dendrimer with a silsesquioxane core) dual modal imaging agent, CLT1-G2-(Gd-DOTA-MA)-Cy5, was synthesized by stepwise conjugation of Gd-DOTA-MA, Cy5 and peptide to the dendrimer. Contrast enhanced MR imaging of the targeted dual imaging agent was evaluated on a Bruker 7T animal scanner with male athymic nude mice bearing orthotopic PC3-GFP prostate tumor. Fluorescence tumor imaging of the agent was carried out on a Maestro fluorescence imaging system. The targeted agent CLT1-G2-(Gd-DOTA-MA)-Cy5 produced greater contrast enhancement in the tumor tissue than the control agent KAREC-G2-(Gd-DOTA-MA)-Cy5 at a dose of 30 μmol-Gd/kg in the MR images of the tumor bearing mice. Signal-to-noise ratio (SNR) of CLT1-G2-(Gd-DOTA-MA)-Cy5 in the tumor tissue was approximately 2 fold of that of the control agent in the first 15 min post-injection. The targeted agent also resulted in bright fluorescence signals in the tumor tissue. The CLT1 peptide targeted nanoglobular dual-imaging agent CLT1-G2-(Gd-DOTA-MA)-Cy5 has a potential for MRI and fluorescence visualization of prostate tumor.

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: A preliminary study

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3–4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB.

Visualizing the prostate gland by MR imaging in young and old mice.

PurposeProstate imaging requires optimization in young and old mouse models. We tested which MR sequences and field strengths best depict the prostate gland in young and old mice; and, whether prostate MR signal, size, and architecture change with age.TechniqueMagnetic resonance imaging (MRI) of the prostate of young (2 months) and old (18 months) male nude mice (n = 6) was performed at 4.7 and 7 T and SCID mice (n = 6) at 7 T field strengths, using T1, fat suppressed T1, DWI, T2, fat suppressed T2, as well as T2-based- and proton density-based Dixon “water only” sequences. Images were ranked for best overall sequence for prostate visualization, prostate delineation, and quality of fat suppression. Prostate volume and signal characteristics were compared and histology was performed.ResultsT2-based-Dixon “water only” images ranked best overall for prostate visualization and delineation as well as fat suppression (n = 6, P<0.001) at both 4.7 T and 7 T in nude and 7T in SCID mice. Evaluated in nude mice, T2-based Dixon “water only” had greater prostate CNR and lower fat SNR at 7 T than 4.7 T (P<0.001). Prostate volume was less in older than younger mice (n = 6, P<0.02 nude mice; n = 6, P<0.002 SCID mice). Prostate T2 FSE as well as proton density-based and T2-based-Dixon “water only” signal intensity was higher in younger than older mice (P<0.001 nude mice; P<0.01 SCID mice) both at 4.7 and 7 T. This corresponded to an increase in glandular hyperplasia in older mice by histology (P<0.01, n = 6).ConclusionT2-based Dixon “water only” images best depict the mouse prostate in young and old nude mice at 4.7 and 7 T. The mouse prostate decreases in size with age. The decrease in T2 and T2-based Dixon “water only” signal with age corresponds with glandular hyperplasia. Findings suggest age should be an important determinant when choosing models of prostate biology and disease.

MicroRNA-31 Activates the RAS Pathway and Functions as an Oncogenic MicroRNA in Human Colorectal Cancer by Repressing RAS p21 GTPase Activating Protein 1 (RASA1)

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

ODM-201, a new generation androgen receptor inhibitor for castration-resistant prostate cancer: Preclinical and phase I data.

65^ Background: Castration resistant prostate cancer (CRPC) is characterized by persistent, high level androgen receptor (AR) expression and resistance to conventional AR inhibitors. ODM-201 is a novel AR inhibitor with unique pharmacologic properties that has shown promising results in preclinical and clinical studies. Methods: AR binding affinity of ODM-201 to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay. Additionally, effects of ODM-201 on the growth of castration resistant VCaP tumors was evaluated. Tumors were established by subcutaneous injection of VCaP prostate cancer cells into male nude mice. After initial tumor growth, mice were castrated. ODM-201 (50 mg/kg QD or BID orally) was initiated upon tumor regrowth. Since the risk of seizures has been reported for some second generation AR inhibitors, ODM-201 concentrations in mouse brain homogenates were studied after repeated oral administration to assess the risk. The clinical effects of ODM-201 (100, 200, 300, 500, 700, 900 mg BID) was examined in a Phase I/II dose-escalation trial in patients (N=24) with progressive metastatic CRPC. Results: ODM-201 binds to wild type AR with superior affinity compared to enzalutamide (Ki of 9 and 39 nM, respectively). In the VCaP CRPC model, ODM-201 significantly (p&lt;0.001 compared to castrated control), and more efficiently than enzalutamide, inhibited tumor growth. After oral administration, tissue/plasma ratio of ODM-201 in mouse brain homogenates was negligible in all studied doses. In the Phase I/II clinical study, ODM-201 (100 to 700 mg results available) was well tolerated, and most commonly reported adverse events were asthenia, diarrhea and nausea. A PSA response (defined as ≥ 50% decrease) was obtained in 13/15 patients evaluable at 12 weeks with all patients achieving partial response or stable disease by RECIST/PCWG2 criteria. Conclusions: ODM-201 is a new generation AR inhibitor with superior preclinical efficacy compared to enzalutamide and bicalutamide, it does not enter the brain in preclinical studies, and it shows very promising activity and no significant toxicity in patients with CRPC. Clinical trial information: NCT01317641.

Abstract C23: Blocking endogenous TBX2 expression abrogates prostate cancer bone metastasis in a xenograft mouse model

Abstract Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human Prostate Cancer (PCa). TBX2, a transcription factor that belongs to the T-box family is known to play critical roles in the embryonic development of the organisms. TBX2 acts as a transcriptional repressor by negatively regulating p21 in humans and p19 ARF in mice. Further, TBX2 has been reported to interact with Retinoblastoma (Rb) and mediates its functional specificity. Previously, TBX2 was found to be over-expressed in BRCA1 and BRCA2 associated breast tumors and melanoma and pancreatic cancer cell lines. Interestingly, a recent report found that TBX2 expression is increased 10-fold at the mRNA level in an androgen-independent bone lesion from a human PCa patient when compared to a bone lesion from an androgen-responsive PCa patient. TBX2 was found to be over-expressed in more aggressive human PCa cell lines and that TBX2 expression correlates with BMP2 expression in these cell lines. Addition of BMP2 to androgen-responsive LAPC4 human prostate cancer cells robustly induces TBX2 expression. Androgen-independent PC3 cells, expressing wild-type TBX2, when infected with the dominant negative TBX2 retroviral construct (PC3-TBX2 DN), display decreased PTHrP, RANKL and SMAD 1,5,8. These results suggest that TBX2, a downstream BMP2 target, csignificantly controls cell signaling network in prostate cancer cells. In vivo, PC3-TBX2 DN cells tagged with firefly luciferase when injected intra-cardiacally in athymic mice lost their ability to metastasize to bone and soft tissues in mice when compared with the control vector infected cells. These results are supported by reduced invasive ability of the PC3-TBX2 DN tumors at the interface between tumor and kidney when grafted under the sub-renal capsule of the male athymic nude mice. Further, utilizing the intra-tibial mouse model, we have found that blocking endogenous TBX2 in PC3 cells significantly reduces their ability to grow in the bone microenvironment. Interestingly, blocking TBX2 does not affect tumor size suggesting that TBX2 is a metastasis suppressor gene. Further, the non-metastatic TrampC2 mouse prostate cancer cells stably over-expressing RANKL show increased Tbx2 expression and develop soft tissue metastases when injected intra-cardiacally in C56/Bl6 immune competent mice. These results suggest that Tbx2 and RANKL pathways cross-talk with each other in prostate cancer metastasis. In summary, we have found that blocking endogenous TBX2 levels in human PCa cells leads to decreased invasion, metastasis and growth in the bone microenvironment, and that TBX2 potentially mediates its effect through BMP2 and RANKL pathways. TBX2, therefore, is a tumor metastasis suppressor and therapeutic target for PCa metastasis. Citation Format: Srinivas Nandana, Manisha Tripathi, Murali Gururajan, Gina Chu, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 expression abrogates prostate cancer bone metastasis in a xenograft mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C23.

Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation

To investigate the role of endoplasmic reticulum (ER) stress in cancer radiotherapy and its molecular mechanism. Tunicamycin (TM) was applied to induce ER stress in human esophageal cancer cell line EC109, and the radiosensitization effects were detected by acute cell death and clonogenic survival assay. Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide. Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase. Autophagic response was determined by acridine orange (AO) staining and Western blotting of microtubule-associated protein-1 light chain-3 (LC3) and autophagy related gene 5 (ATG5). In order to test the biological function of autophagy, specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy, and its effect on cell apoptosis was thus detected. Additionally, involvement of the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway was also detected by Western blotting. Finally, male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations. Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation (IR), which suggested an obvious radiosensitization effect of TM. Moreover, TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. We also observed an increase of AO positive cells, and the protein level of LC3-II and ATG5 was induced by TM treatment, which suggested an autophagic response in EC109 cells. However, inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability, which suggested a cytoprotective role of autophagy in stressed EC109 cells. Furthermore, TM treatment also activated mTORC1, and in turn reduced Akt phosphorylation, which suggested the PI3K/Akt/mTOR signal pathway was involved in the TM-induced autophagic response in EC109 cells. Tumor xenograft results also showed synergistic retarded tumor growth by TM treatment and IR, as well as the involvement of the PI3K/Akt/mTOR pathway. Our data showed that TM treatment sensitized human esophageal cancer cells to radiation via apoptosis and autophagy both in vitro and in vivo.