PC-3 Prostate Cancer Xenografts Research Articles

Lonidamine Induced Selective Acidification and De-Energization of Prostate Cancer Xenografts: Enhanced Tumor Response to Radiation Therapy.

Prostate cancer is a multi-focal disease that can be treated using surgery, radiation, androgen deprivation, and chemotherapy, depending on its presentation. Standard dose-escalated radiation therapy (RT) in the range of 70-80 Gray (GY) is a standard treatment option for prostate cancer. It could be used at different phases of the disease (e.g., as the only primary treatment when the cancer is confined to the prostate gland, combined with other therapies, or as an adjuvant treatment after surgery). Unfortunately, RT for prostate cancer is associated with gastro-intestinal and genitourinary toxicity. We have previously reported that the metabolic modulator lonidamine (LND) produces cancer sensitization through tumor acidification and de-energization in diverse neoplasms. We hypothesized that LND could allow lower RT doses by producing the same effect in prostate cancer, thus reducing the detrimental side effects associated with RT. Using the Seahorse XFe96 and YSI 2300 Stat Plus analyzers, we corroborated the expected LND-induced intracellular acidification and de-energization of isolated human prostate cancer cells using the PC3 cell line. These results were substantiated by non-invasive 31P magnetic resonance spectroscopy (MRS), studying PC3 prostate cancer xenografts treated with LND (100 mg/kg, i.p.). In addition, we found that LND significantly increased tumor lactate levels in the xenografts using 1H MRS non-invasively. Subsequently, LND was combined with radiation therapy in a growth delay experiment, where we found that 150 µM LND followed by 4 GY RT produced a significant growth delay in PC3 prostate cancer xenografts, compared to either control, LND, or RT alone. We conclude that the metabolic modulator LND radio-sensitizes experimental prostate cancer models, allowing the use of lower radiation doses and diminishing the potential side effects of RT. These results suggest the possible clinical translation of LND as a radio-sensitizer in patients with prostate cancer.

Biological Activities of Aurones: A Brief Summary

Abstract: Aurones are structural isomers of flavones and flavonols with the basic C6–C3–C6 skeleton arranged as (Z)-2-benzylidenebenzofuran-3(2H)-one, which contain an exocyclic carbon-carbon double bond bridging the benzofuranone and phenyl rings. In aurone, a chalcone-like group is closed into a 5-membered ring instead of the 6-membered ring more typical of flavonoids, which forms the core for a family of derivatives that are known collectively as aurones. As a kind of flavonoids, aurones are widely distributed in many plants which provide yellow color to some popular ornamental flowers. For a long time aurones had not got enough attention, while in recent years, finally this chemical is coming into researchers' view. As the secondary metabolite in the family of flavonoids, aurones displayed various biological activities, including antioxidant, antiparasitic, antitumor, antiviral, antibacterial, anti-inflammatory, anti-SARS-CoV-2 and neuropharmacological activities. Therefore, aurones have attracted the attention of more and more chemists and pharmaceutical chemists, who realized that it is possible to get lead compounds with better activities via structural modifications of aurones. In some research works, aurone and its derivatives have exhibited good activity, e.g., Xie discovered the heterocyclic variant of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)- one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, and absence of appreciable toxicity, which proved that aurones are valuable compounds worthy of further study. Herein, the biological activities of aurone derivatives are reviewed, which covers the literature since 2000, in which the strategies to develop bioactive aurone derivatives and the structureactivity relationship are highlighted.

Ultrasound-stimulated microbubbles enhancement of fractionated radiation for tumor treatment.

Radiation therapy (XRT) causes numerous biological changes in tumor microenvironment. Radiation vascular response, due to endothelial disruption, can influence treatment outcomes in a dose-dependent manner. Ultrasound-stimulated microbubbles (USMB) have also been demonstrated to create a vascular response in the tumor microenvironment and enhance tumor response when used in combination with XRT. Single doses of 8-10Gy are known to induce activation of acid sphingomyelinase (ASMase)-induced ceramide production, causing vascular damage. Destruction of vasculature results in endothelial apoptosis followed by tumor cell death. The effect of tumor response is known to be synergistic by 10-fold higher cell kill observed when USMB is combined with radiation. In this study, we used an USMB approach in combination with conventional low dose fractionated radiation to enhance endothelial cell responses to XRT in human PC3 prostate cancer xenograft model. Mice were divided into untreated, USMB therapy, fractionated XRT, and combined USMB therapy followed by XRT (USMB + XRT) groups. USMB therapy was delivered twice per week in the USMB-alone and combined USMB + XRT treatment groups over four weeks. Radiation treatments were delivered in fractions of 2Gy/day (total 40Gy in 20 fractions, BED10 = 48Gy) in the XRT-alone and combined USMB + XRT groups. The treatment outcome was evaluated using histopathology, power Doppler, and immunohistochemistry assays. Tumor growth assessment showed that sizes of tumors increased in the control and the single treatment groups over a treatment period of four weeks, but significantly decreased with the combined treatments of USMB + XRT. Immunohistochemical analysis indicated a statistically significant vascular disruption in mice that received treatment involving a full 4-week schedule of combined (USMB + XRT) treatments. A statistically significant increase in vascular disruption was demonstrated through CD68 and trichrome fibrosis staining. Changes in local perfusion assessed using high-frequency power Doppler imaging demonstrated attenuated blood flow in the combined group. This work demonstrates the efficacy of using USMB as a radiation sensitizer in a mouse model of human PC3 tumor xenograft. This radiation treatment enhancement modality has the advantage of targeting tumor vasculature with ultrasound stimulation that can be implemented prior to radiation treatment.

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI>50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [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 465.

Abstract 1043: Advances in cystine-dense peptide (CDP) screening and therapeutic applications

Abstract Cystine-dense peptides (CDPs) are a class of drug-like miniproteins that marry many of the advantages of biologics (high affinity and specificity) and small molecule therapeutics (high tissue permeability and low immunogenicity). The beneficial properties of CDPs, and miniproteins in general, have driven interest in therapeutic applications. However, CDP diversity is vast from every clade of life, and properly interrogating “CDP space” requires specialized screening and modeling tools. With this in mind, we have created an optimized mammalian surface display platform to screen for CDPs of clinical interest using libraries of structurally-diverse native scaffolds optimized for stability. These native CDPs can be structurally modeled, which we did in determining the structures of over 4200 native CDPs. This modeling permits further selection in silico as well as targeted mutagenesis for favorable target-binding capabilities. Hits from these screens are routinely matured to sub-nM affinity. These CDPs can play numerous roles in a drug design pipeline, from an independent drug candidate to a delivery agent for tissue-targeting to a module in a polyspecific biologic. Recent novel CDP candidates have shown promise in immune-oncology space as part of a bispecific T-cell engager targeting PD-L1, where a single 2-week treatment was capable of eliminating subcutaneous PC3 prostate cancer xenograft tumors in 27/30 mice. Besides bispecifics, future directions for the platform include exploring targeted protein degradation. Additionally, we are expanding upon our previous work on CDPs to explore CNS or tumor delivery of therapeutic cargo. The versatility of CDPs and novel screening tools to rapidly identify and mature candidates of interest can facilitate rapid advancement of CDP therapeutics to address difficult targets in oncology. Citation Format: Zachary R. Crook, Emily J. Girard, Gregory P. Sevilla, Mi-Youn Brusniak, Peter B. Rupert, Della J. Friend, Mesfin M. Gewe, Midori Clarke, Ida Lin, Raymond Ruff, Doan Phi, Ashok Bandaranayake, Colin E. Correnti, Andrew J. Mhyre, Natalie W. Nairn, Roland K. Strong, James M. Olson. Advances in cystine-dense peptide (CDP) screening and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1043.

Abstract 2649: Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers

Abstract Purpose: Pro-angiogenic and immune cells expressing chemokine receptor CXCR4 traffic along concentration gradients of the chemokine ligand CXCL12, which disseminates from stromal niches. The CXCR4/CXCL12 axis is hijacked by various cancer types characterized by dramatic CXCR4 and/or CXCL12 upregulation. This chemokine network misregulation causes hyperactivation of CXCR4-mediated processes, leading to (1) excessive stimulation of protective tumor-stromal interactions, (2) metastatic expansion of CXCR4+ cancer cells to CXCL12-rich sites, and (3) intratumoral infiltration of CXCR4+ immunosuppressive cells. Accordingly, CXCR4 antagonists have significant therapeutic potential against cancer. While clinical CXCR4 inhibitors are severely limited by suboptimal pharmacokinetic (PK) properties and off-target activities, several of our tetrahydroisoquinoline-based CXCR4 antagonists exhibit superior selectivity and PK profiles to these clinical comparators (e.g., X4P-001). Methods: To test the hypothesis that improved PK profiles would translate to enhanced efficacy, our lead CXCR4 antagonist EMU-116 was compared to X4P-001 head-to-head in three mouse models of genitourinary cancers. First, human 786.0 renal cell carcinoma (RCC) xenograft-bearing female nude mice were treated p.o., q.d. with vehicle, axitinib (30 mg/kg), X4P-001 (100 mg/kg), EMU-116 (3, 10, or 30 mg/kg), X4P + axitinib, or EMU-116 + axitinib. Subcutaneous tumor volume was monitored using calipers. Second, nude male mice bearing intratibial, luciferase-expressing human PC-3 prostate cancer xenografts were treated with vehicle (p.o., q.d.), docetaxel (10 mg/kg i.p. weekly), X4P-001 (10 or 30 mg/kg p.o., q.d.), EMU-116 (10 or 30 mg/kg p.o., q.d.), X4P + axitinib, or EMU + axitinib. Intratibial tumor volume was monitored via luminescence imaging. Third, syngeneic RENCA RCC tumor-bearing female Balb/c mice were treated p.o., q.d. with vehicle, X4P-001 (30 mg/kg), or EMU-116 (30 mg/kg). Subcutaneous tumor volume was monitored using calipers, and immune cell subsets in bone marrow, blood, tumor, and tumor-draining lymph nodes were quantified via flow cytometry. Results: In the RCC xenograft model in combination with axitinib, EMU-116 (3 or 10 mg/kg) was equally effective at decreasing tumor burden compared to X4P-001 (100 mg/kg), whereas EMU-116 (30 mg/kg) was more effective. In the bone metastatic prostate cancer xenograft model, EMU-116 was as or more effective than X4P-001 when paired with docetaxel. In the syngeneic RCC model, EMU-116 mobilized T cells more effectively than X4P-001. Conclusion: EMU-116, at the same or lower dose, was more efficacious than X4P-001 in these models. These results highlight EMU-116 as a clinical candidate with significant therapeutic potential to synergize with chemotherapeutics, targeted therapies, and immuno-oncology agents. Citation Format: Eric J. Miller, Carrie Q. Sun, Petra Gregorova, Edgars Jecs, Yesim Altas Tahirovic, Robert J. Wilson, Huy H. Nguyen, Savita K. Sharma, Perry Bartsch, Zachary Sticher, Levi Moellering, Priscilla Davidson, Ryan Jajosky, Michael D'Erasmo, Manohar Saindane, Zafer Sahin, Nicholas S. Akins, Alexander A. Kolykhalov, Lawrence Wilson, Rebecca S. Arnold, John A. Petros, Haydn Kissick, Lingjie Xu, Yi Jiang, Dennis C. Liotta. Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2649.

Clickable Radiocomplexes With Trivalent Radiometals for Cancer Theranostics: In vitro and in vivo Studies.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.

Autophagy Induction by α-Santalol in Human Prostate Cancer Cells

Previous studies have shown that the sandalwood oil constituent α-santalol inhibits growth of cultured human prostate cancer cells in vitro and PC-3 prostate cancer xenografts. Along with the studies from our laboratory, it is well established that α-santalol targets the phosphatidylinositol-4,5-bisphosphate 3-kinase-AKT serine/ threonine kinase 1 (AKT) pathway to induce apoptosis but its growth-suppressive effects have not been fully elucidated. The current study was undertaken to investigate the role of autophagy in α-santalol-induced prostate cancer cell death. Cell lines LNCaP and PC-3 were maintained in an atmosphere of 95% air and 5% CO2 at 37°C. Trypan blue dye exclusion assay was employed to assess the effects of α-santalol with/without 3-methyl adenine on the cell viability of prostate cancer cells. Acidic vesicular organelles induced by α-santalol treatment were detected by staining with acridine orange. Immunofluorescence and immunoblotting were performed to analyze expression of proteins involved in the AKT-mammalian target of rapamycin (mTOR) pathway. LNCaP and PC-3 cells upon treatment with α-santalol resulted in characteristic features analogous to autophagic response, including formation of acidic vesicular organelles, recruitment and cleavage of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes. Alpha-santalol treatment further suppressed phosphorylation of activated AKT and mTOR, which are critical regulators of autophagic response. In addition, pre-treatment of PC-3 cells with specific inhibitor of autophagy (3-methyladenine) and co-treatment with α-santalol attenuated the expression of LC3-II and phospho-AKT, and significantly reduced the cell viability. The present study indicates that α-santalol induces autophagy by targeting the AKT-mTOR pathway in prostate cancer cells, which may serve as a protective mechanism.

Comparison of biological properties of [177 Lu]Lu-ProBOMB1 and [177 Lu]Lu-NeoBOMB1 for GRPR targeting.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer and other solid malignancies. Following up on our work on [68 Ga]Ga-ProBOMB1 that had better imaging characteristics than [68 Ga]Ga-NeoBOMB1, we investigated the effects of substituting 68 Ga for 177 Lu to determine if the resulting radiopharmaceuticals could be used with a therapeutic aim. We radiolabeled the bombesin antagonist ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH2 ) with lutetium-177 and compared it with [177 Lu]Lu-NeoBOMB1 (obtained in 54.2 ± 16.5% isolated radiochemical yield with >96% radiochemical purity and 440.8 ± 165.1 GBq/μmol molar activity) for GRPR targeting. Lu-NeoBOMB1 had better binding affinity for GRPR than Lu-ProBOMB1 (Ki values: 2.26 ± 0.24 and 30.2 ± 3.23nM). [177 Lu]Lu-ProBOMB1 was obtained in 53.7 ± 5.4% decay-corrected radiochemical yield with 444.2 ± 193.2 GBq/μmol molar activity and >95% radiochemical purity. In PC-3 prostate cancer xenograft mice, tumor uptake of [177 Lu]Lu-ProBOMB1 was 3.38 ± 1.00, 1.32 ± 0.24, and 0.31 ± 0.04%ID/g at 1, 4, and 24 hours pi. However, the uptake in tumor was lower than [177 Lu]Lu-NeoBOMB1 at all time points. [177 Lu]Lu-ProBOMB1 was inferior to [177 Lu]Lu-NeoBOMB1, which had better therapeutic index for the organs receiving the highest doses.

S179D Prolactin Sensitizes Human PC3 Prostate Cancer Xenografts to Anti-tumor Effects of Well-Tolerated Doses of Calcitriol.

Calcitriol has been shown to have multiple anti-prostate cancer effects both in vitro and in xenograft models, and associations between low levels of calcitriol and more aggressive forms of prostate cancer have been observed clinically. However, the concentrations of calcitriol required to have a substantive anti-cancer effect in vivo are toxic. In previous work, we had observed that the selective prolactin receptor modulator, S179D PRL, sensitized prostate cancer cells in vitro to physiological concentrations of calcitriol through an ability to increase expression of the vitamin D receptor. Here, we have investigated whether administration of S179D PRL would likewise sensitize androgen-insensitive human PC3 xenografts in vivo and do so without inducing tissue damage akin to hypervitaminosis D. Using low concentrations of both S179D PRL (250 ng/h) and calcitriol (up to 220 pg/h), we found no effect of each alone or in combination on the growth rate of tumors. However, there was increased central tumor death with their combination that was more than additive at 250 ng S179D PRL and 220 pg calcitriol per hour. Both S179D PRL and calcitriol alone were antiangiogenic, but their antiangiogenic effects were not additive. Also, both S179D PRL and calcitriol alone increased the number of apoptotic cells in tumor sections, but their combination reduced the number, suggesting more effective clearance of apoptotic cells. Histopathology of the livers and kidneys showed no changes consistent with hypervitaminosis D. We conclude that dual therapy holds promise as a means to harness the anti-tumor effects of well-tolerated doses of calcitriol.

OP#55 - Trastuzumab co-treatment improves survival of mice with PC-3 prostate cancer xenografts treated with [177Lu]Lu-DOTAGA-PEG2-RM26 GRPR antagonists

OP#55 - Trastuzumab co-treatment improves survival of mice with PC-3 prostate cancer xenografts treated with [177Lu]Lu-DOTAGA-PEG2-RM26 GRPR antagonists

Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Positron Emission Tomography Imaging of the Gastrin-Releasing Peptide Receptor with a Novel Bombesin Analogue.

The gastrin-releasing peptide receptor (GRPR), a G protein-coupled receptor, is overexpressed in solid malignancies and particularly in prostate cancer. We synthesized a novel bombesin derivative, [68Ga]Ga-ProBOMB1, evaluated its pharmacokinetics and potential to image GRPR expression with positron emission tomography (PET), and compared it with [68Ga]Ga-NeoBOMB1. ProBOMB1 (DOTA-pABzA-DIG-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ(CH2N)-Pro-NH2) was synthesized by solid-phase peptide synthesis. The polyaminocarboxylate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminal and separated from the GRPR-targeting sequence by a p-aminomethylaniline-diglycolic acid (pABzA-DIG) linker. The binding affinity to GRPR was determined using a cell-based competition assay, whereas the agonist/antagonist property was determined with a calcium efflux assay. ProBOMB1 was radiolabeled with 68GaCl3. PET imaging and biodistribution studies were performed in male immunocompromised mice bearing PC-3 prostate cancer xenografts. Blocking experiments were performed with coinjection of [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14). Dosimetry calculations were performed with OLINDA software. ProBOMB1 and the nonradioactive Ga-ProBOMB were obtained in 1.1 and 67% yield, respectively. The Ki value of Ga-ProBOMB1 for GRPR was 3.97 ± 0.76 nM. Ga-ProBOMB1 behaved as an antagonist for GRPR. [68Ga]Ga-ProBOMB1 was obtained in 48.2 ± 10.9% decay-corrected radiochemical yield with 121 ± 46.9 GBq/μmol molar activity and >95% radiochemical purity. Imaging/biodistribution studies showed that the excretion of [68Ga]Ga-ProBOMB1 was primarily through the renal pathway. At 1 h postinjection (p.i.), PC-3 tumor xenografts were clearly delineated in PET images with excellent contrast. The tumor uptake for [68Ga]Ga-ProBOMB1 was 8.17 ± 2.57 percent injected dose per gram (% ID/g) and 9.83 ± 1.48% ID/g for [68Ga]Ga-NeoBOMB1, based on biodistribution studies at 1 h p.i. This corresponded to tumor-to-blood and tumor-to-muscle uptake ratios of 20.6 ± 6.79 and 106 ± 57.7 for [68Ga]Ga-ProBOMB1 and 8.38 ± 0.78 and 39.0 ± 12.6 for [68Ga]Ga-NeoBOMB1, respectively. Blockade with [d-Phe6,Leu-NHEt13,des-Met14]bombesin(6-14) significantly reduced the average uptake of [68Ga]Ga-ProBOMB1 in tumors by 62%. The total absorbed dose was lower for [68Ga]Ga-ProBOMB1 in all organs except for bladder compared with [68Ga]Ga-NeoBOMB1. Our data suggest that [68Ga]Ga-ProBOMB1 is an excellent radiotracer for imaging GRPR expression with PET. [68Ga]Ga-ProBOMB1 achieved a similar uptake as [68Ga]Ga-NeoBOMB1 in tumors, with enhanced contrast and lower whole-body absorbed dose.

No effect of unacylated ghrelin administration on subcutaneous PC3 xenograft growth or metabolic parameters in a Rag1-/- mouse model of metabolic dysfunction.

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome.

Limited short-term effects on human prostate cancer xenograft growth and epidermal growth factor receptor gene expression by the ghrelin receptor antagonist [D-Lys3]-GHRP-6.

The ghrelin axis regulates many physiological functions (including appetite, metabolism, and energy balance) and plays a role in disease processes. As ghrelin stimulates prostate cancer proliferation, the ghrelin receptor antagonist [D-Lys3]-GHRP-6 is a potential treatment for castrate-resistant prostate cancer and for preventing the metabolic consequences of androgen-targeted therapies. We therefore explored the effect of [D-Lys3]-GHRP-6 on PC3 prostate cancer xenograft growth. NOD/SCID mice with PC3 prostate cancer xenografts were administered 20 nmoles/mouse [D-Lys3]-GHRP-6 daily by intraperitoneal injection for 14 days and tumour volume and weight were measured. RNA sequencing of tumours was conducted to investigate expression changes following [D-Lys3]-GHRP-6 treatment. A second experiment, extending treatment time to 18 days and including a higher dose of [D-Lys3]-GHRP-6 (200 nmoles/mouse/day), was undertaken to ensure repeatability. We demonstrate here that daily intraperitoneal injection of 20 nmoles/mouse [D-Lys3]-GHRP-6 reduces PC3 prostate cancer xenograft tumour volume and weight in NOD/SCID mice at two weeks post treatment initiation. RNA-sequencing revealed reduced expression of epidermal growth factor receptor (EGFR) in these tumours. Further experiments demonstrated that the effects of [D-Lys3]-GHRP-6 are transitory and lost after 18 days of treatment. We show that [D-Lys3]-GHRP-6 has transitory effects on prostate xenograft tumours in mice, which rapidly develop an apparent resistance to the antagonist. Although further studies on [D-Lys3]-GHRP-6 are warranted, we suggest that daily treatment with the antagonist is not a suitable treatment for advanced prostate cancer.

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma

BackgroundAKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition.MethodsCellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS.ResultsIn vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model.ConclusionThis MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Abstract 2674: Novel tubulin polymerization inhibitors repress tumor xenografts in nude mice and leukemia in zebrafish

Abstract In an effort to screen natural compounds and their derivatives as novel agents for cancer treatment, we developed two semisynthetic nature compounds, A14 and A52, which have potencies in an in vitro cell proliferation studies against LS174T and PC-3 cells in the mid-nanomolar range. A14 inhibited more than 90% of in vitro cancer cell proliferation around 300 nM and also inhibited in vivo PC-3 prostate cancer xenografts in nude mice. Cell morphology studies and comparisons with known antineoplastic agents suggested that A14 disrupted microtubule organization. In vitro tubulin polymerization assay suggested that A14 inhibited this process. Based on molecular docking, A14 fits very well in the colchicine-binding site at the interface between the alpha-tubulin and beta-tubulin. Further analyses suggest that A14 sensitivity may correlate with CDK/cyclin activity. A14 inhibited PC-3 prostate cancer xenografts in nude mice. In addition to solid tumors, A14 and its analog A52 strongly inhibited a panel of human leukemia cancer cell lines. Furthermore, these compounds inhibited Myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model. Although many tubulin inhibitors were available, there is no compound that targeting the colchicine-binding site has been approved by FDA for cancer treatment. This new family of tubulin polymerization inhibitors could be further developed for the treatment of both solid and liquid tumors. Citation Format: Yanqi Xie, Liliia Kril, Wen Zhang, Mykhaylo Frasinyuk, Svitlana Bondarenko, Elizabeth Hausman, Zachary Martin, Przemyslaw Wyrebek, Tianxin Yu, Changguo Zhan, Vitaliy Sviripa, Jessica Blackburn, David Watt, Chunming Liu. Novel tubulin polymerization inhibitors repress tumor xenografts in nude mice and leukemia in zebrafish [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 2674.

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.

MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited prostate cancer cell growth by inducing apoptosis through the targeting of phosphatidylinositol 3‐kinase‐Akt and mitogen‐activated protein kinase survival pathways in androgen‐responsive transformed human prostate cancer epithelial cell lines. However, the preventive effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment using complementary PC‐3 prostate cancer cell culture and xenograft models. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 protein and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrated that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells which was complimented by siRNA‐HSP40 knock‐down that also inhibited cell migration and growth. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored as a novel therapeutic in castration resistant prostate cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

Bombesin receptor 2 (BB2) and integrin αvβ3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB2- and integrin αvβ3-targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD2-BBN heterotrimer, composed of (7-14)BBN-NH2 peptide (BBN) linked to the E[ c(RGDyK)]2 dimer peptide (RGD2), bearing the new linker type [Pro-Gly]12. The heterodimer E[c(RGDyK)]2-PEG3-Glu-(Pro-Gly)12-BBN(7-14)-NH2 (RGD2-PG12-BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good αvβ3 affinities and high BB2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( Ki = 24 nM). 64Cu-DOTA and 64Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB2 and integrin αvβ3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.