Prostate Tumor Xenografts In Mice Research Articles

Translational Development of a Zr-89-Labeled Inhibitor of Prostate-specific Membrane Antigen for PET Imaging in Prostate Cancer

PurposeWe present here a Zr-89-labeled inhibitor of prostate-specific membrane antigen (PSMA) as a complement to the already established F-18- or Ga-68-ligands.ProceduresThe precursor PSMA-DFO (ABX) was used for Zr-89-labeling. This is not an antibody, but a peptide analogue of the precursor for the production of [177Lu]Lu-PSMA-617. The ligand [89Zr]Zr-PSMA-DFO was compared with [68Ga]Ga-PSMA-11 and [18F]F-JK-PSMA-7 in vitro by determination of the Kd value, cellular uptake, internalization in LNCaP cells, biodistribution studies with LNCaP prostate tumor xenografts in mice, and in vivo by small-animal PET imaging in LNCaP tumor mouse models. A first-in-human PET was performed with [89Zr]Zr-PSMA-DFO on a patient presenting with a biochemical recurrence after brachytherapy and an ambiguous intraprostatic finding with [18F]F-JK-PSMA-7 but histologically benign cells in a prostate biopsy 7 months previously.Results[89Zr]Zr-PSMA-DFO was prepared with a radiochemical purity ≥ 99.9% and a very high in vitro stability for up to 7 days at 37 °C. All radiotracers showed similar specific cellular binding and internalization, in vitro and comparable tumor uptake in biodistribution experiments during the first 5 h. The [89Zr]Zr-PSMA-DFO achieved significantly higher tumor/background ratios in LNCaP tumor xenografts (tumor/blood: 309 ± 89, tumor/muscle: 450 ± 38) after 24 h than [68Ga]Ga-PSMA-11 (tumor/blood: 112 ± 57, tumor/muscle: 58 ± 36) or [18F]F-JK-PSMA-7 (tumor/blood: 175 ± 30, tumor/muscle: 114 ± 14) after 4 h (p < 0.01). Small-animal PET imaging demonstrated in vivo that tumor visualization with [89Zr]Zr-PSMA-DFO is comparable to [68Ga]Ga-PSMA-11 or [18F]F-JK-PSMA-7 at early time points (1 h p.i.) and that PET scans up to 48 h p.i. clearly visualized the tumor at late time points. A late [89Zr]Zr-PSMA-DFO PET scan on a patient with biochemical recurrence (BCR) had demonstrated intensive tracer accumulation in the right (SUVmax 13.25, 48 h p.i.) and in the left prostate lobe (SUV max 9.47), a repeat biopsy revealed cancer cells on both sides.Conclusion[89Zr]Zr-PSMA-DFO is a promising PSMA PET tracer for detection of tumor areas with lower PSMA expression and thus warrants further clinical evaluation.

R1 Regulates Prostate Tumor Growth and Progression By Transcriptional Suppression of the E3 Ligase HUWE1 to Stabilize c-Myc.

Prostate cancer is a prevalent public health problem, especially because noncutaneous advanced malignant forms significantly affect the lifespan and quality of life of men worldwide. New therapeutic targets and approaches are urgently needed. The current study reports elevated expression of R1 (CDCA7L/RAM2/JPO2), a c-Myc-interacting protein and transcription factor, in human prostate cancer tissue specimens. In a clinical cohort, high R1 expression is associated with disease recurrence and decreased patient survival. Overexpression and knockdown of R1 in human prostate cancer cells indicate that R1 induces cell proliferation and colony formation. Moreover, silencing R1 dramatically reduces the growth of prostate tumor xenografts in mice. Mechanistically, R1 increases c-Myc protein stability by inhibiting ubiquitination and proteolysis through transcriptional suppression of HUWE1, a c-Myc-targeting E3 ligase, via direct interaction with a binding element in the promoter. Moreover, transcriptional repression is supported by a negative coexpression correlation between R1 and HUWE1 in a prostate cancer clinical dataset. Collectively, these findings, for the first time, characterize the contribution of R1 to prostate cancer pathogenesis. IMPLICATIONS: These findings provide evidence that R1 is a novel regulator of prostate tumor growth by stabilizing c-Myc protein, meriting further investigation of its therapeutic and prognostic potential.

DHX15 promotes prostate cancer progression by stimulating Siah2-mediated ubiquitination of androgen receptor.

Androgen receptor (AR) activation is critical for prostate cancer development and progression, including castration-resistance. The nuclear export signal of AR (NESAR) plays an important role in AR intracellular trafficking and proteasome-dependent degradation. Here, we identified the RNA helicase DHX15 as a novel AR co-activator using a yeast mutagenesis screen and revealed that DHX15 regulates AR activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity. DHX15 and Siah2 form a complex with AR, through NESAR. DHX15 stabilized Siah2 and enhanced its E3 ubiquitin ligase activity, resulting in AR activation. Importantly, DHX15 was upregulated in prostate cancer specimens and its expression was correlated with Gleason scores and PSA recurrence. Furthermore, DHX15 immunostaining correlated with Siah2. Finally, DHX15 knockdown inhibited the growth of C4-2 prostate tumor xenografts in mice. Collectively, our data argue that DHX15 enhances AR transcriptional activity and contributes to prostate cancer progression through Siah2.

Abstract 3343: Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer

Abstract Cancer stem cells (CSCs) represent the most tumorigenic, metastatic and therapy resistant cell subpopulation within human tumors. Current therapies target bulk tumor cells while spare the most aggressive CSC subpopulation. Understanding the mechanisms responsible for the acquisition and maintenance of the CSC phenotype will help to identify new strategies to target CSCs. In this study, we describe a link between deregulated expression of the ETS transcription factor ESE3/EHF and upregulation of Lin28A and Lin28B in prostate CSC-enriched subpopulations. Furthermore, using various cell line models and in vitro/in vivo experimental systems we demonstrate the efficacy of targeting the Lin28A/B axis for selective elimination of cancer cells with tumor-initiating and stem-like properties. Mechanistically, we found that ESE3/EHF represses transcription of Lin28A and Lin28B in normal prostate epithelial cells. Downregulation of ESE3/EHF in prostate tumors led to upregulation of Lin28A and Lin28B and consequent reduction of microRNAs (miRNAs) of the let-7 family, which exert tumor suppressor functions. These events promoted cell transformation and expansion of the prostate CSC subpopulation. Conversely, targeting Lin28A/Lin28B with small interfering RNAs (siRNAs) in transformed prostate epithelial cells and prostate cancer cell lines restored the levels of let-7 miRNAs, decreased expression of several CSC marker genes, and restrained tumor-sphere formation and self-renewal properties in vitro and tumor-initiating capability in vivo. Notably, systemic treatment with a siRNA targeting Lin28B reduced growth of prostate tumor xenografts in mice. This was associated with a significant contraction of the CSC subpopulation in tumor xenografts, as demonstrated by the reduced content of ex vivo tumor-sphere forming cells, reduced expression of CSC marker genes, and upregulation of let-7 miRNAs. Furthermore, tumor cells derived from siLin28B-treated xenografts exhibited reduced in vivo tumor-initiating and self-renewal capability, in line with a persistent loss of CSC properties. Collectively, these data establish the Lin28/let-7 axis as a critical element in malignant transformation and acquisition of tumor-initiating and stem-like properties and identify a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Citation Format: Domenico Albino, Gianluca Civenni, Cecilia Dallavalle, Martina Roos, Hartmut Jahns, Jonathan Hall, Giuseppina M. Carbone, Carlo V. Catapano. Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3343.

Abstract 366: Translational modeling of docetaxel-thalidomide combination treatment in metastatic, castrate-resistant prostate cancer: predicting clinical response using preclinical data

Abstract A major cause of drug failure in the clinic is that preclinical studies do not predict with sufficient certainty what will happen in clinical practice. Accurately translating information from animal studies to the clinic would have a major impact on attrition rate. We have developed a mathematical model of a tumor cell population called the Virtual Tumour, which has been used extensively to predict the efficacy of single drug or drug combination treatment in preclinical studies. We have now extended and adapted our preclinical model to predict efficacy in the clinic, thus creating the “Virtual Tumour Clinical.” Here we show a comparative study of the preclinical Virtual Tumour calibrated model for prostate tumor xenografts in mice, with a Virtual Tumour Clinical version calibrated with a clinical data set comprising 53 prostate cancer patients treated with thalidomide, 25 treated with docetaxel and 50 treated with a docetaxel and thalidomide combination. PSA measurements were used as proxy for tumor size. We analysed the consistency, the capability and the limitations of the models in translating the effect of the drug combination from the preclinical situation to the clinic. Citation Format: Eric Fernandez, Hitesh Mistry, Frances Brightman, David Orrell, William L. Dahut, William D. Figg, Wilfried D. Stein, Christophe Chassagnole. Translational modeling of docetaxel-thalidomide combination treatment in metastatic, castrate-resistant prostate cancer: predicting clinical response using preclinical data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2014-366

A Rac1/Cdc42 GTPase-Specific Small Molecule Inhibitor Suppresses Growth of Primary Human Prostate Cancer Xenografts and Prolongs Survival in Mice

Deregulated Rho GTPases Rac1 and Cdc42 have been discovered in various tumors, including prostate and Rac protein expression significantly increases in prostate cancer. The Rac and Cdc42 pathways promote the uncontrolled proliferation, invasion and metastatic properties of human cancer cells. We synthesized the novel compound AZA1 based on structural information of the known Rac1 inhibitor NSC23766. In the current study we investigated the effects of inhibition of these pathways by AZA1 on prostate tumorigenicity by performing preclinical studies using a xenograft mouse model of prostate cancer. In androgen-independent prostate cancer cells, AZA1 inhibited both Rac1 and Cdc42 but not RhoA GTPase activity in a dose-dependent manner and blocked cellular migration and proliferation. Cyclin D1 expression significantly decreased following Rac1/Cdc42 inhibition in prostate cancer cells. AZA1 treatment also down-regulated PAK and AKT activity in prostate cancer cells, associated with induction of the pro-apoptotic function of BAD by suppression of serine-112 phosphorylation. Daily systemic administration of AZA1 for 2 weeks reduced growth of human 22Rv1 prostate tumor xenografts in mice and improved the survival of tumor-bearing animals significantly. These data suggest a role of AZA1 in blocking Rac1/Cdc42-dependent cell cycle progression, cancer cell migration and increase of cancer cell apoptosis involving down-regulation of the AKT and PAK signaling pathway in prostate cancer cells. We therefore propose that a small-molecule inhibitor therapy targeting Rac1/Cdc42 Rho GTPase signaling pathways may be used as a novel treatment for patients with advanced prostate cancer.

1272 POTENTIAL IMMUNOTHERAPEUTIC APPLICATIONS OF PHOSPHODIESTERASE INHIBITION FOR PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111272 POTENTIAL IMMUNOTHERAPEUTIC APPLICATIONS OF PHOSPHODIESTERASE INHIBITION FOR PROSTATE CANCER David Siemens, Tom Hamilton, Klodiana Gjoncaj, Nianping Hu, Erin Bell, and Charles Graham David SiemensDavid Siemens Kingston, Canada More articles by this author , Tom HamiltonTom Hamilton Kingston, Canada More articles by this author , Klodiana GjoncajKlodiana Gjoncaj Kingston, Canada More articles by this author , Nianping HuNianping Hu Kingston, Canada More articles by this author , Erin BellErin Bell Kingston, Canada More articles by this author , and Charles GrahamCharles Graham Kingston, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.958AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The cyclic nucleotide phosphodiesterases (PDEs) are a family of enzymes that play a central role in controlling cyclic nucleotide (cGMP) action and previous studies have ascribed a protective role of cGMP mediated signaling on hypoxia- mediated cancer progression. Herein we describe the expression levels of the known PDE variants in prostate cancer as well as explore the functional role that they play in cancer immune escape. METHODS cGMP phosphodiesterase assays were used to measure the PDE activity in human prostate cancer cell lines DU145 and PC3. Western Blot analysis was performed to determine the presence of the PDEs in human tissue samples. The effect of PDE inhibition on hypoxia-mediated immune escape mechanisms was determined after pre-incubating cells in 0.5% or 20% O2 in the absence or presence of Zaprinast (10-7–10-6 M) and subsequent fow cytometric analysis of MICA, an essential tumor-associated antigen required for innate immune responses. A NK-competent murine model was used to measure the in vivo effects of Zaprinast on the growth of human prostate cancer xenografts. RESULTS PDE activity assays indicated that the majority of cGMP PDE activity in prostate cancer cell lines is made up from a combination of PDE5 and PDE11. The same two PDE variants also predominated in human malignant prostate tissue as detected using Western blot. TUNEL assays revealed some increased apoptosis when prostate cells were treated with a PDE inhibitor in both hypoxic and standard oxygen conditions. MICA expression was reduced when prostate cancer cells were exposed to hypoxia; however, immunogenicity could be restored by incubating hypoxic samples with a PDE inhibitor (see figure, p<0.01). Finally, growth of human prostate tumor xenografts in mice was inhibited by PDE inhibition compared to controls (p<0.05). CONCLUSIONS PDE 5 and 11 are present in human prostate cancer tissue and contribute to the majority of cGMP PDE activity in prostate cancer cell lines. Inhibition of PDE activity, reestablishing cGMP cell signaling in the hypoxic tumor environment, would appear to have beneficial effects including modulating hypoxia-induced cancer immune escape. These results indicate that PDE inhibition may represent a novel therapeutic or adjuvant target for men with prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e509 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information David Siemens Kingston, Canada More articles by this author Tom Hamilton Kingston, Canada More articles by this author Klodiana Gjoncaj Kingston, Canada More articles by this author Nianping Hu Kingston, Canada More articles by this author Erin Bell Kingston, Canada More articles by this author Charles Graham Kingston, Canada More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

A 19F-NMR approach using reporter molecule pairs to assess beta-galactosidase in human xenograft tumors in vivo.

Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection in the target tissue and the longevity of gene expression. Thus, there is increasing interest in the development of non-invasive in vivo reporter techniques to assay gene expression. We recently demonstrated the ability to detect beta-galactosidase activity in stably transfected human prostate tumor xenografts in mice in vivo using 19F NMR. We now extend the studies to human MCF7 breast cancer cells growing as xenografts in nude mice. Moreover, by using two spectrally resolved reporters (o-fluoro-p-nitrophenyl-beta-D-galactopyranoside and an isomer), two tumors could be interrogated simultaneously revealing lacZ transgene activity in a stably transfected tumor versus no activity in a wild-type tumor. Most significantly, hydrolytic activity observed by 19F NMR corresponded to differential activity in lacZ-expressing tumors.

Downregulation of BCL-2 Induces Downregulation of Carbonic Anhydrase IX, Vascular Endothelial Growth Factor, and pAkt and Induces Radiation Sensitization

Our group previously demonstrated that expression of the oncogene, Bcl-2, was associated with radiation resistance. The aim of the present study was to determine whether Bcl-2 expression in radiation-resistant tumors was associated with expression of carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), and pAkt and whether downregulation of Bcl-2 could modulate the expression of CAIX, VEGF, and pAkt. Two prostate cancer cell lines, PC-3-Bcl-2 and PC-3-Neo, were injected into the subcutaneous flanks of 192 athymic male nude mice; 96 received PC-3 Bcl-2 and 96 PC-3-Neo. The mice were then treated with antisense Bcl-2 oligodeoxynucleotide (ASODN), reverse control ODN, or vehicle only (mock treatment) with or without irradiation (4 Gy). The tumors were monitored for growth (ie, volume) over time, resected at various points, and processed and sectioned for protein analyses. Bcl-2 ASODN treatment was associated with downregulation of Bcl-2, VEGF, pAkt, and CAIX. PC-3-Bcl-2 and PC-3-Neo prostate tumor xenografts in mice treated with the combination of Bcl-2 ASODN and irradiation were significantly (ie, one third) smaller than those in mice treated with reverse control ODN alone, Bcl-2 ASODN alone, irradiation alone, or reverse control ODN plus irradiation (P = 0.0001). An increased tumor response to the combined therapy was associated with decreased expression of Bcl-2, VEGF, and pAkt proteins. These findings have demonstrated an intimate relationship among CAIX, VEGF, pAkt, and Bcl-2 in prostate tumors. Thus, CAIX might prove effective as a potential marker of tumor radiation resistance. Downregulation of CAIX might lead to radiation sensitization. Consequently, the combination of CAIX reduction and radiotherapy warrants further consideration as a new strategy for therapy.

19 F‐NMR detection of lacZ gene expression via the enzymic hydrolysis of 2‐fluoro‐4‐nitrophenyl β‐D‐galactopyranoside in vivo in PC3 prostate tumor xenografts in the mouse 1

Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding beta-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing beta-galactosidase (beta-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl beta-D-galactopyranoside (OFPNPG) is readily hydrolyzed by beta-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach.

A peptidomimetic inhibitor of ras functionality markedly suppresses growth of human prostate tumor xenografts in mice. Prospects for long-term clinical utility.

These studies sought to evaluate the antitumor properties of an inhibitor of ras functionality, L-744,832, which acts at the level of its associated protein farnesyltransferase. Studies were carried out to measure the effects of L-744,832 alone and in combination with paclitaxel (PTXL) against TSU-PR1, DU-145 and PC-3 human prostate tumors xenografted to NCR-nul (AT) mice. Tumor-bearing mice were treated on a schedule of daily for 5 days x2 or 3 with the MTD of L-744,832 and every 3-4 days x4 with the MTD of PTXL starting 3-5 days after tumor implantation. Tumor volume in millimeters (4/3pir3) was measured 3 5 days after cessation of treatment and the increase in tumor volume in treated and control groups compared. Statistical analysis was carried out by the Chi-squared test. L-744,832 at its MTD markedly inhibited the growth of all three tumors (TIC for increase in tumor mass varied from 11% to 15% and inhibition of growth had a rapid onset (within 1-2 days) and was independent of ras gene status. Estimated tumor doubling times were 8-12-fold greater in treated animals than in control animals. Treatment with L-744,832 for as long as 3 weeks had no untoward effects on the mice as determined by gross examination or necropsy. Administration of L-744,832 with this same dose and schedule potentiated the growth-inhibitory effect of PTXL at its MTD and induced some regression of TSU-PR1 with no obvious deleterious effects on the mice. L-744,832 could be safely administered over a protracted period of time to mice at doses which were markedly inhibitory to the growth of three human prostate tumor xenografts and in combination with PTXL was also well tolerated and brought about some regression of the TSU-PR1 tumor. Overall, these results suggest that L-744,832 could be clinically useful for long-term treatment of early-stage prostate cancer in patients and as an adjunct to cytotoxic therapy for late stages of this disease.