LNCaP Prostate Cancer Xenograft Research Articles

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Abstract Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Lolke de Haan, Ian Kirby, Narinder Janghra, Veronica Gil, Pedro Alves, Kristina Zaitseva, Meghann Kerr, Ben Leatherdale, Afroze Patel, Marie Thoelke, Shiran Huang, John A Hartley, Patrick H van Berkel. Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2638.

Improve the Biodistribution with Bulky and Lipophilic Modification Strategies on Lys-Urea-Glu-Based PSMA-Targeting Radiotracers.

Since prostate-specific membrane antigen (PSMA) is upregulated in nearly all stages of prostate cancer (PCa), PSMA can be considered a viable diagnostic biomarker and treatment target in PCa. In this study, we have developed five 68Ga-labeled PSMA-targeted tracers, 68Ga-Flu-1, 68Ga-Flu-2, 68Ga-9-Ant, 68Ga-1-Nal, and 68Ga-1-Noi, to investigate the effect of lipophilic bulky groups on the pharmacokinetics of PSMA inhibitors compared to 68Ga-PSMA-11 and then explore their in vitro and in vivo properties. 68Ga-labeled PSMA inhibitors were obtained in 88.53-99.98% radiochemical purity and at the highest specific activity of up to 20 MBq/μg. These compounds revealed a highly efficient uptake and internalization into LNCaP cells and increased over time. PET imaging and biodistribution studies were performed in mice bearing PSMA expressing LNCaP prostate cancer xenografts. All tracers enabled clear visualization of tumors in PET images with excellent tumor-to-background contrast. The biodistribution studies showed that all these radioligands were excreted mainly via the renal pathway. The in vivo biodistribution of 68Ga-Flu-1 revealed higher tumor uptake (40.11 ± 9.24 %ID/g at 2 h p.i.) compared to 68Ga-PSMA-11 (28.10 ± 5.96 %ID/g at 2 h p.i.). Both in vitro and in vivo experiments showed that chemical modification of the lysine fragment significantly impacts tumor-targeting and pharmacokinetic properties. Great potential to serve as new PET tracers for prostate cancer has been revealed with these radiotracers─68Ga-Flu-1 in particular.

In vivo evaluation of nanostructured lipid carrier systems (NLCs) in mice bearing prostate cancer tumours

Nanostructure lipid carriers (NLCs) were developed for the delivery of curmumin (CRN), a potent anticancer agent with low bioavailability, for the treatment of prostate cancer. NLCs prepared using high pressure homogenization (HPH) with around 150 nm particle size, − 40 V ζ-potential and excellent long-term stability. Cellular uptake of CRN-SLN showed nanoparticle localization in the cytoplasm around the nucleus. CRN-NLCs were assessed using flow cytometry and found to cause early and late apoptotic events at 100 μg/ml CRN concentrations. CRN-NLC nanoparticles were administrated to nude mice with LNCaP prostate cancer xenografts and demonstrated substantial tumour volume suppression (40%) with no weight loss compared to pure CRN (ethanolic solution). Overall, NLCs were proved a suitable carrier for passive drug delivery and cancer treatment.Graphical abstract

Abstract 1392: Synergistic antitumor efficacy of radium-223 in combination with enzalutamide in the intratibial LNCaP prostate cancer xenograft model

Abstract Radium-223 dichloride (Ra-223) is a targeted alpha therapy that binds to newly formed bone matrix in bone metastases and induces DNA double-strand breaks in cancer cells, osteoblasts and osteoclasts. It is used for treating men with castration-resistant prostate cancer (CRPC) and bone metastases. Enzalutamide is a second-generation androgen receptor inhibitor also used for treating the same patient population, and the combination of Ra-223 and enzalutamide is currently being investigated in clinical trials. We evaluated the antitumor efficacy of Ra-223 and enzalutamide in the LNCaP intratibial model mimicking prostate cancer metastasized to bone. LNCaP prostate cancer cells were inoculated into the right tibia of male NOD.scid mice. The mice were randomized (n=9/group) based on serum PSA and treated with vehicle, Ra-223 (330 kBq/kg, i.v., Q4W x 2), enzalutamide (30 mg/kg, p.o., QD) or with a combination treatment of Ra-223 and enzalutamide, for 28 days. Serum PSA levels were analyzed at the end of the study and compared to the pre-treatment levels. Serum bone formation and resorption biomarkers, PINP and CTX, respectively, were measured during the study. Tumor-induced abnormal bone area, Ra-223 uptake and bone formation were determined by X-ray, gamma counter and histomorphometry, respectively. The healthy tibiae were evaluated by microCT. Combination treatment showed synergistic antitumor efficacy as observed by lower PSA levels when compared to the vehicle, Ra-223 or enzalutamide monotherapies (p=0.04, p=0.008 and p=0.002, respectively). A statistical interaction between Ra-223 and enzalutamide treatments was found (p=0.003), confirming the synergistic effect. In combination treatment, the serum PSA change relative to pre-treatment levels was 18% of the vehicle. Accordingly, a decreasing trend (p=0.08) in tumor-induced abnormal bone changes was associated with the combination treatment in the tumor-bearing tibiae (46% of the vehicle), whereas no changes in total bone structure were observed in the healthy tibiae. The serum levels of PINP and CTX were lowest in mice treated with the combination treatment. Compared to Ra-223 monotherapy, concurrent administration of enzalutamide with Ra-223 did not affect either Ra-223 uptake or bone formation rate in tumor-bearing or healthy tibiae, respectively. Compared to Ra-223 and enzalutamide monotherapies, the combination treatment demonstrated synergistic antitumor efficacy by decreasing PSA levels in the LNCaP intratibial model. Despite of prominent effects on tumor growth, the combination treatment was not observed to compromise bone health in the healthy tibiae. In conclusion, these preclinical results support the ongoing phase 3 trials PEACE III (NCT02194842) & ESCALATE (NCT04237584) of this combination. Citation Format: Mari I. Suominen, Matias Knuuttila, Jukka Vääräniemi, Birgitta Sjöholm, Esa Alhoniemi, Dominik Mumberg, Sanna-Maria Käkönen, Arne Scholz. Synergistic antitumor efficacy of radium-223 in combination with enzalutamide in the intratibial LNCaP prostate cancer xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1392.

Synergistic antitumor efficacy of radium-223 and enzalutamide in the intratibial LNCaP prostate cancer xenograft model.

105 Background: Radium-223 dichloride (Ra-223) is a targeted alpha therapy that binds to newly formed bone matrix in bone metastases and induces DNA double-strand breaks in cancer cells, osteoblasts and osteoclasts. It is used for treating men with castration-resistant prostate cancer (CRPC) and bone metastases. Enzalutamide (enza) is a second-generation androgen receptor inhibitor also used for treating the same patient population, and the combination of Ra-223 and enza is currently being investigated in clinical trials. We evaluated the antitumor efficacy of Ra-223 and enza in the LNCaP intratibial model mimicking prostate cancer metastasized to bone. Previously, additive or synergistic antitumor effects were not observed when Ra-223 was combined with abiraterone and prednisone in the LNCaP model (Suominen et al., AACR 2020). Methods: LNCaP prostate cancer cells were inoculated into the right tibia of male NOD.scid mice. The mice were randomized (n = 9/group) based on serum PSA and treated with vehicle, Ra-223 (330 kBq/kg, i.v., Q4W x 2), enza (30 mg/kg, p.o., QD) or with a combination treatment of Ra-223 and enza, for 28 days. Serum PSA levels were analyzed at the end of the study and compared to the pre-treatment levels. Serum bone formation and resorption biomarkers, PINP and CTX, respectively, were measured during the study. Tumor-induced abnormal bone area and Ra-223 uptake were determined by X-ray and gamma counter, respectively. The healthy tibiae were evaluated by microCT. Results: Combination treatment showed synergistic antitumor efficacy as observed by lower PSA levels when compared to the vehicle, Ra-223 or enza monotherapies (p = 0.04, p = 0.008 and p = 0.002, respectively). A statistical interaction between Ra-223 and enza treatments was found (p = 0.003), confirming the synergistic effect. In combination treatment, the serum PSA change relative to pre-treatment levels was 18% of the vehicle. Accordingly, a decreasing trend (p = 0.08) in tumor-induced abnormal bone changes was associated with the combination treatment in the tumor-bearing tibiae (46% of the vehicle), whereas no changes in total bone structure/quality were observed in the healthy tibiae. Compared to monotherapies, the combination treatment had the most prominent lowering effect on the bone metabolism biomarkers PINP and CTX during the study. Concurrent administration of enza with Ra-223 did not affect Ra-223 uptake in tumor-bearing tibiae. Conclusions: Compared to Ra-223 and enza monotherapies, the combination treatment demonstrated synergistic antitumor efficacy by decreasing PSA levels in the LNCaP intratibial model. Despite of prominent effects on tumor growth, the combination treatment was not observed to compromise bone health in the healthy tibiae. In conclusion, these preclinical results support the ongoing phase 3 trials PEACE III (NCT02194842) & ESCALATE (NCT04237584) of this combination.

Abstract 2452: Dysregulated expression of the human long noncoding RNA GHSROS may influence prostate cancer progression and resistance to docetaxel

Abstract Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated with metastasis and poor prognosis. Finally, we developed two distinct antisense oligonucleotides (ASOs) targeting GHSROS, achieving >60% knockdown, and their function was assessed in vitro. ASO inhibition of GHSROS expression reciprocally regulated cell growth and migration and the expression of a range of genes. These ASOs are currently being assessed in preclinical animal models. Our findings suggest that the long noncoding RNA GHSROS reprograms prostate cancer cells toward a more aggressive phenotype and that the lncRNA represents a promising therapeutic target. Citation Format: Patrick B. Thomas, Penny L. Jeffery, Gahete D. Manuel, Eliza J. Whiteside, Michelle Maugham, Carina Walpole, Jennifer H. Gunter, Elizabeth D. Williams, Colleen C. Nelson, Adrian C. Herington, Raul M. Luque, Rakesh N. Veedu, Lisa K. Chopin, Inge Seim. Dysregulated expression of the human long noncoding RNA GHSROS may influence prostate cancer progression and resistance to docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2452.

Effects of Linker Modification on Tumor-to-Kidney Contrast of 68Ga-Labeled PSMA-Targeted Imaging Probes.

68Ga-PSMA-11 is currently the most popular prostate-specific membrane antigen (PSMA) radioligand used in the clinic to detect prostate cancer and metastases. However, the high uptake of 68Ga-PSMA-11 in kidneys can create halo-artifacts resulting in lower detection sensitivity for lesions adjacent to the kidneys. In this study, we developed two 68Ga-labeled PSMA-targeted tracers, 68Ga-HTK01166 and 68Ga-HTK01167, based on 68Ga-PSMA-617 with the goal of improving tumor-to-kidney ratio compared to 68Ga-PSMA-11. The 2-naphthylalanine (2-Nal) in PSMA-617 was replaced with 2-indanylglycine (Igl) or 3,3-diphenylalanine (Dip) to synthesize HTK01166 and HTK01167, respectively. Binding affinities ( Ki) of Ga-PSMA-11, Ga-PSMA-617, Ga-HTK01166, and Ga-HTK01167 to PSMA were 3.13 ± 0.40, 1.23 ± 0.08, 5.74 ± 2.48, and 25.7 ± 9.84 nM, respectively, as determined by in vitro competition binding assays. 68Ga labeling was performed in HEPES buffer with microwave heating, and 68Ga-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were obtained in 46-69% average decay-corrected radiochemical yield with >99% radiochemical purity and 62.9-152 GBq/μmol average specific activity. PET imaging and biodistribution studies were performed in mice bearing PSMA-expressing LNCap prostate cancer xenografts. All tracers enabled clear visualization of tumors in PET images with excellent tumor-to-background contrast. The uptake values (%ID/g) for tumor and kidneys at 1 h postinjection were 8.91 ± 0.86 and 204 ± 70.6 for 68Ga-PSMA-11, 16.7 ± 2.30 and 29.2 ± 5.14 for 68Ga-PSMA-617, 14.1 ± 4.40 and 147 ± 59.6 for 68Ga-HTK01166, and 7.79 ± 1.65 and 4.30 ± 1.80 for 68Ga-HTK01167. The tumor-to-kidney ratios for 68Ga-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were 0.05 ± 0.02, 0.63 ± 0.10, 0.10 ± 0.02, and 1.98 ± 0.63, respectively. Compared with 68Ga-PSMA-617, 68Ga-HTK01166 showed comparable tumor uptake and almost 5-fold higher kidney uptake, whereas 68Ga-HTK01167 exhibited lower tumor and kidney uptake. Compared with 68Ga-PSMA-11, 68Ga-HTK01167 had similar tumor uptake and tumor-to-blood contrast ratio (23.8 ± 6.71 vs 20.4 ± 4.98) but higher tumor-to-background contrast ratios for other background organs especially for kidneys. Our data indicate that substitution of 2-Nal in PSMA-617 with other lipophilic amino acid can modulate PSMA binding affinity and their pharmacokinetics in vivo.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs–s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs– with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs– showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs– activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs–, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs– in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with 18F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. 18F-CJ-042794 was synthesized via a copper-mediated 18F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5±1.1% (n=2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that 18F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12±0.08%ID/g (n=5) uptake value and moderate tumour-to-muscle contrast ratio (2.73±0.22) at 1h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of 18F-CJ-042794 for PET imaging of EP4 receptor expression in cancer.

Abstract 3490: The effect of novel CYP17 inhibitor galeterone on gonadal and tumor progestogen and androgen levels in SCID mice bearing LNCaP prostate cancer xenografts

Abstract Prostate cancer growth is driven by androgen-dependent activation of the androgen receptor (AR). Inhibition of CYP17A1, a cytochrome P450 enzyme responsible for the conversion of progestogens to androgens, is a key strategy in the treatment of prostate cancer. CYP17A1 has both 17α-hydroxylase and 17,20-lyase function. The 17α-hydroxylase catalyzes the conversion of progesterone/pregnenolone to 17α-hydroxyprogesterone/17α-hydroxypregnenolone and the 17,20-lyase converts 17α-hydroxyprogesterone/17α-hydroxypregnenolone to androstenedione (AD)/dehydroepiandrosterone (DHEA). Galeterone, a novel AR degrader and antagonist, is also an inhibitor of CYP17A1 and has been shown to inhibit the growth of prostate cancer cells and tumors. However, the effects of galeterone on steroid levels affected by CYP17A1 inhibition have not been examined in vivo. Male SCID mice bearing LNCaP tumors were randomized to receive vehicle (30% beta-cyclodextran in saline) or 0.15mmol/kg galeterone (either po or sc). Mice were treated twice daily, seven days a week. Route of administration did not alter the efficacy of galeterone, which significantly reduced tumor volume (p = 0.044 and p = 0.049, sc, po). Upon completion of the experiment, tumors, testes, and plasma were collected for analysis of steroid levels. Analysis of intratumoral steroids showed an increase in progesterone (1.7- and 2-fold, sc, po) and a decrease in AD (89% and 77% reduction, sc, po). Similar results were observed in levels of pregnenolone which increased (1.4- and 3.0-fold, sc, po) while DHEA decreased (72% and 17% reduction, sc, po). Interestingly, levels of 17α-hydroxyprenenolone were increased in the sc treatment arm, suggesting selectivity of galeterone for the lyase over the hydroxylase catalytic function. Both routes of administration reduced intratumoral testosterone (97% and 77% reduction, sc, po). Intratesticular androgen levels showed similar trends compared to those in tumors. Following galeterone treatment, the levels of androgen precursors were higher in the testes than in the tumor (pregnenolone increased 2.3- and 1.5-fold sc, po; progesterone elevated 6.4- and 12.8-fold sc, po). Plasma androgen levels varied from those observed in the tumor and testes. However, galeterone treatment reduced the levels of AD (98% and 68% reduction, sc, po) and testosterone (99.6% and 99% reduction, sc, po). Together, these results show for the first time that galeterone specifically targets CYP17A1 in vivo as demonstrated by reduction of its enzymatic products DHEA, AD and testosterone. In addition, a selectivity for the lyase function was observed, as evidenced by greater decreases in DHEA and AD than in 17α-hydroxypregenelone. This is consistent with findings that patients treated with galeterone do not require cortisol replacement therapy and do not show symptoms of mineralocorticoid excess. Citation Format: Amanda J. Schech, Gauri J. Sabnis, Stephen Yu, Vincent C.O. Njar, Douglas B. Jacoby, Peter Nelson, Ruth Dumpit, Angela M.H. Brodie. The effect of novel CYP17 inhibitor galeterone on gonadal and tumor progestogen and androgen levels in SCID mice bearing LNCaP prostate cancer xenografts. [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 3490.

Oral simvastatin administration delays castration-resistant progression and reduces intratumoral steroidogenesis of LNCaP prostate cancer xenografts.

Growing evidence supports the idea that de novo steroidogenesis has an important role in prostate cancer's progression to the castration-resistant state following androgen deprivation therapy. Therefore, reducing the availability of cholesterol for use as a precursor in androgen synthesis may reduce proliferation and disease progression. LNCaP xenograft-bearing mice were castrated and administered simvastatin via diet, and tumor volume and PSA concentration were monitored for 8 weeks post castration. Levels of serum and intratumoral androgens along with serum simvastatin and common toxicity markers were measured at end point. Reduced post-castration tumor growth rate in simvastatin-treated mice correlated with delayed time to castration-resistant progression, determined by two serum PSA doublings from post-castration nadir, when compared with xenografts in mice on control diet. At 8 weeks post castration, serum simvastatin levels were comparable to clinically relevant human doses with no evidence of overt muscle or liver toxicity. This suppressed post-castration tumor growth in the simvastatin diet group was correlated with reduced intratumoral testosterone and dihydrotestosterone levels. Reduced tumor growth and intratumoral androgen levels observed in simvastatin-treated, castrated mice harboring LNCaP xenograft suggests that suppressing de novo steroidogenesis can delay castration-resistant progression of this tumor model.

Methylselenocysteine preventing castration-resistant progression of prostate cancer.

Castration-resistant progression of prostate cancer after androgen deprivation therapy remains a critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor activity is an established driver of castration-resistant progression, and upregulation of androgen receptor expression has been implicated to contribute to the resurgent androgen receptor activity. We reported previously that methylselenocysteine can decrease the expression and activity of androgen receptor. Here we investigated the ability of methylselenocysteine to inhibit castration-resistant progression of prostate cancer. The regrowth of LNCaP prostate cancer xenografts after castration was monitored. The levels of prostate-specific antigen in mouse serum were measured by ELISA. Tumor cell proliferation and apoptosis were analyzed via Ki-67 immunohistochemistry and TUNEL assay, respectively. Intratumoral angiogenesis was assessed by immunohistochemistry staining of vascular endothelial growth factor and CD31. We showed that methylselenocysteine delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation. This was accompanied by decreased serum levels of prostate-specific antigen, inhibition of prostate cancer cell proliferation and tumor angiogenesis, as well as downregulation of androgen receptor and induction of apoptosis in the relapsed tumors. The present study represents the first to show the preclinical efficacy of methylselenocysteine in delaying castration-resistant progression of prostate cancer. The findings provide a rationale for evaluating the clinical application of combining methylselenocysteine with androgen deprivation therapy for the treatment of advanced prostate cancer.

Preparation of Asymmetric Urea Derivatives that Target Prostate-Specific Membrane Antigen for SPECT Imaging

Prostate-specific membrane antigen (PSMA) has been identified as a diagnostic and therapeutic target for prostate cancer. (S)-2-[3-[(R)-1-Carboxy-2-mercaptoethyl]ureido-pentanedioic acid (Cys-CO-Glu) were used to design novel PSMA targeting probes by nucleophilic conjugate addition between cysteine and maleimide based reagents. 3 ([(123)I]IGLCE) was synthesized by this strategy and showed high affinity for PSMA. Results of binding inhibition assays of these derivatives suggested the importance of an aromatic group and succinimide moiety for high affinity. [(123)I]3 was evaluated in vivo with PSMA positive LNCaP and PSMA negative PC-3 human prostate cancer xenograft bearing mice. [(125)I]3 accumulated in LNCaP tumors but not in PC-3 tumors, and the accumulation was inhibited by 2-(phosphonomethyl)pentanedioic acid (2-PMPA). Use of [(123)I]3 provided positive images of LNCaP tumors in single photon emission tomography scans. These results warrant further evaluation of [(123)I]3 and its derivatives as radiolabeled probes for the diagnosis of prostate cancer.

Abstract 4952: A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling.

Abstract Hedgehog (Hh) signaling is a highly conserved inter- and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. While TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologues of the Hh-activated genes, Gli1, Gli2, and Ptch1, was substantially suppressed, while expression of the corresponding human orthologues was unaffected. Since androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression. Citation Format: Mazyar Ghaffari, Naokazu Ibuki, Irene Iu, Mitali Pandey, Ladan Fazli, Masahide Kashiwagi, Hideaki Tojo, Osamu Nakanishi, Martin E. Gleave, Michael E. Cox. A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling. [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 4952. doi:10.1158/1538-7445.AM2013-4952

Ansprechen auf eine Docetaxel-therapie im LNCaP-Prostata-karzinom-Xenograft-Mausmodell

SummaryThe aim of this study was to determine whether [11C]choline can be used for docetaxel therapy response assessment in a LNCaPprostate cancer xenograft mouse model using [11C]choline small-animal PET/CT. Animals, methods: The androgen-dependent human prostate cancer cell line LNCaP was implanted subcutaneously into the left flanks of 17 SCID-mice, 12.5 mg testosterone platelets were implanted in the neck wrinkle. All mice were injected 4–6 weeks after xenograft implantation with 37 MBq [11C]choline via the tail vein. Dynamic imaging was performed for 60 minutes with a small-animal PET/CT scanner. After the first [11C]choline PET/CT imaging 8 mice were subsequently injected intravenously with docetaxel twice (days 1 and 5) at a dose of 3 mg/kg body weight. 8 mice were treated with PBS as a control. [11C]choline PET/CT imaging was performed on day 7, 14 and 21 after treatment. Image analysis was performed using tumor/ muscle (T/M) ratios (ROIT/ROIM = T/M ratio). Results: All LNCaP tumours could be visualized by [11C]choline PET/CT. Before treatment the mean T/M ratio was 2.0 ± 0.2 in the docetaxel-treated group and 1.9 ± 0.2 in the control group (p = 0.837). There was a reduction in the mean [11C]choline uptake after docetaxel treatment of the tumours of the LNCaP cell line as early as 1 week after initiation of therapy (T/Mmean ratio 1.5 ± 0.2 after one week, 1.3 ± 0.2 after 2 weeks and 1.4 ± 0.2 after 3 weeks). There was no decrease in [11C]choline uptake in the control group. Conclusion: Our results show that [11C]choline has the potential for use in the early monitoring of the therapeutic effect of docetaxel in a LNCaP prostate cancer xenograft animal model.

Activity of oral VT-464, a selective CYP17-lyase inhibitor, in the LNCaP prostate cancer xenograft.

4671 Background: With the FDA approval of abiraterone acetate, inhibition of CYP17 (17α hydroxylase/C17, 20-lyase) is now a validated approach to the treatment of castration-resistant prostate cancer. VT-464 is a novel, selective CYP17-lyase inhibitor with decreased activity against CYP17 hydroxylase (less mineralcocorticoid and glucocorticoid effects). The study objectives were to observe the effects of VT-464 in a prostate cancer xenograft model and to compare its activity to abiraterone acetate and surgical castration. Methods: SCID mice were implanted subcutaneously with LNCaP cells. When tumors reached 100 mm3, mice were randomized to receive vehicle (0.5% CMC in saline, 5 mL/kg), VT-464 at 15, 50, or 100 mg/kg p.o. b.i.d. A second cohort of LNCaP tumor-bearing mice received vehicle, surgical castration, or VT-464, or abiraterone acetate at 100 mg/kg p.o. b.i.d. for 28 days. Terminal blood and tumor concentrations were analyzed on day 28, four hours after the last dose. Results: In the first LNCaP xenograft cohort, percent growth inhibition (± S.E.) of 9.6 (±15.6), 38.5 (±12.4), and 73.9 (±13.2) was observed on day 21 of treatment for VT-464 doses of 15, 50, and 100 mg/kg, respectively. Growth reduction at 100 mg/kg was statistically significant compared to vehicle control from day 7 to 28. VT-464 was well tolerated with insignificant weight loss at all doses. In the second cohort, VT-464-treated (100 mg/kg) mice had significantly reduced tumor volumes on day 28 compared to control and abiraterone acetate (p<0.05, p<0.01, respectively). Reduction in tumor volumes were similar between VT-464-treated (100 mg/kg) and castrate animals. Plasma and tumor analyses revealed much greater plasma and tumor exposure of VT-464 compared to abiraterone acetate. Conclusions: VT-464 exhibited dose-dependent growth inhibition with significantly reduced tumor volumes at the highest dose compared to abiraterone acetate. The activity in VT-464-treated animals was similar to that of castrate animals. These preclinical results show promising activity of VT-464 in the treatment of prostate cancer.

Abstract 4827: Humanized hepsin neutralizing antibody RO5486055 inhibits tumor growth and leads to accumulation of hepsin substrate laminin-332

Abstract Hepsin is overexpressed in prostate and other cancers where it is implicated in promoting tumor growth, invasion, and metastasis. To further our understanding of the role of hepsin in prostate and breast cancer, a fully humanized monoclonal antibody that recognizes human and cynomolgus hepsin has been developed. RO5486055 selectively binds to hepsin but not related type II transmembrane serine proteases, and neutralizes hepsin serine protease activity with an IC50 in the single digit nM range. In LNCaP prostate cancer cells, both shRNA knockdown of the hepsin gene and RO5486055 treatment cause a similar accumulation of the β3 chain of laminin-332, a known hepsin substrate. With ip administration in mice, RO5486055 demonstrates dose-dependent exposure and a long serum half-life of 168-406 hours. RO5486055 attenuates tumor growth in the LNCaP prostate cancer and T-47D breast cancer xenograft models, but not in the CRW-22Rv1 prostate or MCF-7 breast cancer xenografts. In these models, it has been shown that the level of the β3 chain of laminin-332 detected by Western blot analysis predicts sensitivity to RO5486055-mediated growth inhibition. Moreover, accumulation of the β3 chain during RO5486055 treatment correlates with antitumor activity. In 8/10 hepsin-expressing patient-derived prostate tumors, an inverse correlation between hepsin and β3 chain expression is observed. The β3 chain of laminin-332 may therefore be useful as both a predictive and response biomarker for anti-hepsin therapy. RO5486055-mediated tumor growth inhibition is enhanced by combination with the EGFR-targeted antibody cetuximab in LNCaP prostate cancer xenografts, and by combination with hormone withdrawal in T-47D breast cancer xenografts. These preclinical results suggest that hepsin-directed therapy could be effective in prostate and breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4827. doi:1538-7445.AM2012-4827

Activity of VT-464, a selective CYP17 lyase inhibitor, in the LNCaP prostate cancer xenograft model.

64 Background: With the recent FDA approval of abiraterone acetate, CYP17 (17α hydroxylase/C17, 20-lyase) has become a proven target for the treatment of castration- resistant prostate cancer. Inhibition of CYP17-lyase causes a decrease in circulating androgens, severely hampering activation of the androgen receptor signaling pathway that prostate cancer relies on for proliferation. However, inhibition of CYP17-hydroxylase, a second enzymatic activity of CYP17, leads to an increase in upstream steroids that can cause mineralocorticoid excess syndrome as well as a decrease in cortisol production. VT-464 is a novel, selective CYP17 lyase inhibitor with decreased activity against CYP17 hydroxylase. The study objectives were to observe the effects of VT-464 in a prostate cancer xenograft model and to compare its activity to abiraterone acetate and surgical castration. Methods: SCID mice were implanted subcutaneously with LNCaP cells. When tumors reached 100mm3, mice were randomized to receive vehicle (0.5% CMC in saline, 5ml/kg), or VT-464 at 15, 50, or 100mg/kg p.o. bid. A second cohort of LNCaP tumor-bearing mice received vehicle, surgical castration, or VT-464 or abiraterone acetate at 100mg/kg p.o. bid for 28 days. Results: In the LNCaP xenograft model, percent growth inhibition (± S.E.) of 9.6 (±15.6), 38.5(±12.4), and 73.9 (±13.2) was observed on day 21 of treatment for the VT-464 doses of 15, 50, and 100 mg/kg, respectively. VT-464 -treated (100mg/kg) mice had a significantly reduced tumor volume ratio (V/V0) on day 28 compared to control and abiraterone acetate (p<0.05, p<0.01, respectively). Reduction in tumor volume ratios were similar between VT-464-treated (100 mg/kg) and castrate animals. Conclusions: VT-464 exhibited dose-dependent growth inhibition with significantly reduced tumor growth at the highest dose compared to abiraterone acetate. The reduction in tumor growth in VT-464-treated animals was similar to that of castrate animals. These preclinical results show promising activity of VT-464 in the treatment of prostate cancer.

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo

The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 μM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing Cmax, while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors.

Abstract A62: Increased histone acetylation, but no inhibition of HDAC activity, by kale sprout intervention in a human prostate cancer xenograft model

Abstract Cruciferous vegetable consumption has been associated with a reduced risk of prostate cancer. These vegetables such as broccoli and kale are rich in glucosinolates, which are converted to isothiocyanates (ITCs) by myrosinase. ITCs have cancer preventive potential through various mechanisms including anti-proliferative effects. ITC metabolites have been recently identified as inhibitors of histone deacetylases (HDAC), which have the potential to turn on epigenetically silenced genes, leading to cell cycle arrest and apoptosis. In the present study we investigated the effect of kale sprouts commercially available as a food supplement to inhibit the growth of human LNCaP prostate cancer cells in a xenograft model. We were interested whether kale sprout intervention would affect histone acetylation and in studying underlining mechanisms, e.g. HDACs and histone acetyltransferases (HAT). LNCaP human prostate cancer cells were injected to male nude Balb/c mice (10 per group). Mice were fed with either regular rodent chow or chow supplemented with 20% kale sprouts (containing about 60 µmol glucosinolates per 5 g chow consumed daily, together with active myrosinase) from a week before cell injection until sacrifice. 7.5 weeks after injection, tumor tissue was collected. Kale sprout intervention did not significantly inhibit tumor growth in the xenograft model. However, global acetylation levels of histone H4 detected by Western blotting were increased in the kale sprout-treated group, whereas acetylated histone H3 levels were not significantly changed. The increase of H4 lysine 5, 8, 12 and 16 acetylation was individually confirmed. Among them, acetylated H4 K12 was most abundant. Even though global acetylation of H3 did not change, acetylated H3K9 was significantly increased. Unexpectedly, HDAC activity in nuclear extracts of tumor tissue determined by using ZMAL substrate was not significantly inhibited in tumor tissue by kale sprout intervention. Also, protein expression of HDAC family members HDAC1-5 was not changed overall, although HDAC 5 expression was reduced while HDAC2 expression was increased. Instead, kale sprout treatment significantly increased mRNA levels of PCAF and CBP quantified by qRT-PCR, by 36% and 23%, respectively, in tumor tissue (p<0.001), which may contribute to increased H4 acetylation. Taken together, our results indicate that dietary consumption of kale sprouts increased global acetylation of histone H4 in LNCaP prostate cancer xenograft model, possibly by induction of HAT expression rather than inhibition of HDAC activity. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A62.