LNCaP Xenografts Research Articles

Diverse effects of phospholipase A2 receptor expression on LNCaP and PC-3 prostate cancer cell growth in vitro and in vivo.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1’s function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor’s pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.

A Single Dose of 225Ac-RPS-074 Induces a Complete Tumor Response in an LNCaP Xenograft Model.

Promising biochemical responses to 225Ac-prostate-specific membrane antigen (PSMA) 617, even in patients who are refractory to β-particle radiation, illustrate the potential of targeted α-therapy for the treatment of metastatic castration-resistant prostate cancer. However, side effects such as xerostomia are severe and irreversible. To fully harness the potential of targeted α-therapy, it is necessary to increase the therapeutic index of the targeted radioligands. One emerging strategy is to increase clearance half-life through enhanced binding to serum albumin. We have evaluated the albumin-binding PSMA-targeting ligand RPS-074 in a LNCaP xenograft model to determine its potential value to the treatment of prostate cancer. Methods:225Ac-RPS-074 was evaluated in male BALB/c mice bearing LNCaP xenograft tumors. A biodistribution study was performed over 21 d to determine the dosimetry in tumors and normal tissue. The dose response was measured in groups of 7 mice using 37, 74, and 148 kBq of 225Ac-RPS-074 and compared with positive and negative control groups. Mice were sacrificed when tumor volume exceeded 1,500 mm3Results:225Ac-RPS-074 was labeled in greater than 98% radiochemical yield and showed high (>10% injected dose/g) and sustained accumulation in LNCaP tumors from 24 h to beyond 14 d. Signal in blood and highly vascularized tissues was evident over the first 24 h after injection and cleared by 7 d. The tumor-to-kidney ratio was 4.3 ± 0.7 at 24 h and 62.2 ± 9.5 at 14 d. A single injection of 148 kBq induced a complete response in 6 of 7 tumors, with no apparent toxic effects. Treatment with 74 kBq induced a partial response in 7 of 7 tumors, but from 42 d, 6 of 7 experienced significant regrowth. The 37-kBq group experienced a survival benefit relative to the negative control but not compared with the positive control group. Conclusion: A single dose of 148 kBq of 225Ac-RPS-074 induced a complete response in 86% of tumors, with tumor-to-normal-tissue ratios that predict an improved therapeutic index. The use of the macropa chelator enabled quantitative radiolabeling and may facilitate the clinical translation of this promising targeted α-therapeutic.

Tracking Tumor Colonization in Xenograft Mouse Models Using Accelerator Mass Spectrometry

Here we introduce an Accelerator Mass Spectrometry (AMS)-based high precision method for quantifying the number of cancer cells that initiate metastatic tumors, in xenograft mice. Quantification of 14C per cell prior to injection into animals, and quantification of 14C in whole organs allows us to extrapolate the number of cancer cells available to initiate metastatic tumors. The 14C labeling was optimized such that 1 cancer cell was detected among 1 million normal cells. We show that ~1–5% of human cancer cells injected into immunodeficient mice form subcutaneous tumors, and even fewer cells initiate metastatic tumors. Comparisons of metastatic site colonization between a highly metastatic (PC3) and a non-metastatic (LnCap) cell line showed that PC3 cells colonize target tissues in greater quantities at 2 weeks post-delivery, and by 12 weeks post-delivery no 14C was detected in LnCap xenografts, suggesting that all metastatic cells were cleared. The 14C-signal correlated with the presence and the severity of metastatic tumors. AMS measurements of 14C-labeled cells provides a highly-sensitive, quantitative assay to experimentally evaluate metastasis and colonization of target tissues in xenograft mouse models. This approach can potentially be used to evaluate tumor aggressiveness and assist in making informed decisions regarding treatment.

O-9 - Network analysis of prostate cancer-derived CWR22, LNCaP and VCAP xenografts for elucidation of the key regulators in castration resistance development

O-9 - Network analysis of prostate cancer-derived CWR22, LNCaP and VCAP xenografts for elucidation of the key regulators in castration resistance development

Synthesis and Preclinical Characterization of the PSMA-Targeted Hybrid Tracer PSMA-I&F for Nuclear and Fluorescence Imaging of Prostate Cancer.

The prostate-specific membrane antigen (PSMA)–targeted radiotracers 68Ga/177Lu-PSMA-I&T and 99mTc-PSMA-I&S (for imaging and surgery) are currently successfully used for clinical PET imaging, radionuclide therapy, and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a PSMA-I&T–based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated. Methods: The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T, and their corresponding natGa-/68Ga- and natLu/177Lu counterparts was determined in LNCaP cells via competitive binding assays (IC50) and dual-tracer radioligand and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17 SCID and LNCaP xenograft–bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of 1 tumor-bearing animal as well as PSMA immunohistochemistry and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed. Results: Compared with the parent PSMA-I&T analogs, the PSMA affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9 < IC50 < 10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94%–95%), and very low accumulation in nontarget organs was observed already at 1 h after injection. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5% ± 1.8 percentage injected dose per gram) and the kidneys (106% ± 23 percentage injected dose per gram). Tumor-to-background ratios of 2.1, 5.2, 9.6, and 9.6 for blood, liver, intestines, and muscle, respectively, at 1 h after injection led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by immunohistochemistry. Conclusion: Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T–based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

Abstract A091: Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer

Abstract Background: Sustained androgen receptor pathway activation is the hallmark of castration-resistant prostate cancer (CRPC). Therapeutic strategies for CRPC include non-ligand-binding domain targeted degradation of androgen receptor (AR) and AR variants (ARV). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that are critical transcriptional regulators of the androgen receptor. We have developed CCS1477, which is a potent, selective, and orally active small-molecule inhibitor of the bromodomain of p300/CBP, and investigated its role in regulating androgen receptor expression and function. Methods: Binding affinity of CCS1477 to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity (proliferation, cell cycle analysis, and biomarker knockdown) was demonstrated in a panel of prostate cells lines representing hormone-responsive (LNCaP), hormone-independent (DU145, PC3), and castration-resistant disease (22Rv1, VCaP, LNCaP-AR). A JQ1 resistant cell line was generated by culturing cells in increasing doses of JQ1 over a period of approximately 9 months and used to differentiate potency and functional activity of BET vs p300/CBP inhibition in vitro. In vivo efficacy, linked to inhibition of biomarkers, was determined in 22Rv1 and LNCaP xenograft models. Results: CCS1477 binds to p300 and CBP with high affinity (KD=1.3/1.7nM) and selectivity (KD=222nM; BRD4). It is a potent inhibitor of cell proliferation in castration-resistant cell lines (IC50= 96nM,22Rv1; 49nM,VCaP; 150nM,LNCaP-AR) with minimal effect in AR-ve lines (PC3, DU145). These inhibitory effects on cell proliferation were accompanied by a decrease in the number of cells in S phase and an increase in the number of cells in the G1/G0 phase of the cell cycle. We have developed a JQ1 resistant 22Rv1 cell line and observed a marked reduction in sensitivity (proliferation end point) to JQ1 as expected, which is accompanied by cross-resistance to a variety of other BET inhibitors, including iBET-151 and OTX-015. In contrast, and as evidence for a differentiated mechanism, sensitivity to CCS1477 is retained in this JQ1 resistant line. Inhibition of the bromodomain of p300/CBP downregulates AR-FL, AR-V7 and c-Myc protein in 22Rv1 cells by Western blot and this is accompanied by profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR. The in vivo PK properties of CCS1477 are consistent with qd or qod oral dosing in mouse. CCS1477 dosed at 10mg/kg qd, 20mg/kg qd, or 30mg/kg qod caused complete tumor growth inhibition over 28 days in a 22Rv1 xenograft model, including extended duration in the absence of the drug for a further 24 days. This was accompanied by complete inhibition of plasma PSA and significant knockdown of tumor AR-FL, AR-V7, and c-Myc protein as well as c-Myc and TMPRSS2 mRNA expression. At 100mg/kg, a single dose of CCS1477 induces expression of cleaved PARP and when dosed orally every three days, causes tumor regression in the 22Rv1 model. Furthermore, CCS1477 results in almost complete tumor growth inhibition in a bicalutamide-resistant LNCaP xenograft model, when given alone or in combination with enzalutamide. Conclusions: Taken together, these data support the concept that therapeutic targeting of the p300/CBP bromodomain results in a novel and differentiated approach to targeting androgen receptor pathway activation in castration-resistant prostate cancer. Citation Format: Nigel Brooks, Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser. Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A091.

Preclinical Evaluation of Discorhabdins in Antiangiogenic and Antitumor Models.

Elements of the hypoxia inducible factor (HIF) transcriptional system, a key regulator of the cellular hypoxic response, are up-regulated in a range of cancer cells. HIF is fundamentally involved in tumor angiogenesis, invasion, and energy metabolism. Inhibition of the transcriptional activity of HIF may be of therapeutic benefit to cancer patients. We recently described the identification of two marine pyrroloiminoquinone alkaloids with potent activity in inhibiting the interaction between the oncogenic transcription factor HIF-1α and the coactivator protein p300. Herein, we present further characterization data for these two screening hits: discorhabdin H (1) and discorhabdin L (2), with a specific focus on their anti-angiogenic and anti-tumor effects. We demonstrated that only discorhabdin L (2) possesses excellent anti-angiogenic activity in inhibiting endothelial cell proliferation and tube formation, as well as decreasing microvessel outgrowth in the ex vivo rat aortic ring assay. We further showed that discorhabdin L (2) significantly inhibits in vivo prostate tumor growth in a LNCaP xenograft model. In conclusion, our findings suggest that discorhabdin L (2) represents a promising HIF-1α inhibitor worthy of further drug development.

Molar Activity of Ga-68 Labeled PSMA Inhibitor Conjugates Determines PET Imaging Results.

Radiopharmaceuticals targeting the enzyme prostate-specific membrane antigen (PSMA; synonyms: glutamate carboxypeptidase II, NAALADase; EC 3.4.17.21) have recently emerged as powerful agents for diagnosis and therapy (theranostics) of prostate carcinoma (PCa). The radiation doses for therapeutic application of such compounds are limited by substantial uptakes in kidneys and salivary glands, with excess doses reportedly leading to radiotoxicity-related adverse effects, such as kidney insufficiency or xenostomia. On the basis of the triazacyclononane-triphosphinate (TRAP) chelator, monomeric to trimeric conjugates of the PSMA inhibitor motif lysine-urea-glutamic acid (KuE) were synthesized by means of Cu(I)-mediated (CuAAC) or 5-aza-dibenzocyclooctyne (DBCO)-driven, strain-promoted click chemistry (SPAAC), which were labeled with gallium-68 for application in positron emission tomography (PET), and characterized in terms of PSMA affinity (determined in cellular displacement assays against I-125-BA) and lipophilicity (expressed as log D). Using subcutaneous murine LNCaP (PSMA-positive human prostate carcinoma) xenografts, the influence of ligand multiplicity, affinity, polarity, and molar activity (i.e., mass dose) on the uptakes in tumor, kidney, salivary, and background (muscle) was analyzed by means of region-of-interest (ROI) based quantification of small-animal PET imaging data. As expected, trimerization of the KuE motif resulted in high PSMA affinities (IC50 ranging from 6.0-1.5 nM). Of all parameters, molar activity/cold mass had the most pronounced influence on PET uptakes. Because accumulation in nontumor tissues was effected to a larger extent than tumor uptakes, lower molar activities resulted in substantially better tumor-to-organ ratios. For example, for one trimer, 68Ga-AhxKuE3 (IC50 = 1.5 ± 0.3 nM, log D = -3.8 ± 0.1), a higher overall amount of active compound (12 pmol vs 2 nmol, equivalent to molar activities of 1200 and 8 MBq/nmol) resulted in a remarkable reduction of the kidney-to-tumor ratio from 11.4 to 1.4, respectively, at 60 min p.i. Our study suggests that, for PSMA-targeting radiopharmaceuticals, molar activity has a more pronounced influence on small-animal PET imaging results than structural or in vitro parameters.

Abstract 5077: Cancer-associated fibroblasts stimulate tumor growth and metastatic spread in an orthotopic prostate cancer xenograft model

Abstract Introduction: In recent years, it has become more and more evident, that the different cell populations comprising the so-called “tumor microenvironment” contribute substantially to the processes of carcinogenesis, local tumor growth and metastatic spread. Especially the tumor-promoting roles of cancer-associated fibroblasts have been the focus of several elaborate in-vitro studies. However, evidence from representative in-vivo models is scarce. In this study, we coinjected prostate cancer (PCa) cells together with different populations of prostate fibroblasts (cancer-associated (CAF), not cancer-associated (NCAF), benign prostate hyperplasia associated (BPHF)) and examined their impact on local tumor growth and the development of metastases. Material &amp; Methods: We isolated fibroblast primary cultures from tumor-bearing (CAF) and tumor-free (NCAF) tissue samples of a radical prostatectomy specimen as well as from tissue samples of a transvesical prostate adenoma enucleation (BPHF). 5x105 CAF/NCAF/BPHF cells were injected into the prostate of SCID mice together with 5x105 LuCaP136 spheroids or LNCaP cells (n=8 for each group). Tumor growth was monitored by high-resolution ultrasonography (hrUS), micro-CT, 9.4T MRI and serum PSA-measurements. Animals were sacrificed after 10 weeks. Results: The coinjection of CAFs fostered tumor growth in LuCaP136 but not in LNCaP xenografts. Primary tumor volumes were significantly higher in LuCaP136+CAF mice compared to LuCaP136+NCAF and LuCaP136+BPHF mice (p&amp;lt;0.01 for each time point). Besides that, LuCaP136+CAF mice had significantly higher serum PSA values than their NCAF and BPHF counterparts (p&amp;lt;0.01 for each time point). Significantly more lymph node metastases (LN mets) were seen in LuCaP136+CAF mice (one mouse without, four mice with 2 and three mice with ≥3 LN mets) compared to LuCaP136+NCAF (one mouse without, four mice with 1 and one mouse with 2 LN mets) and LuCaP136+BPHF mice (two mice without, three mice with 1 and two mice with 2 LN mets) (p&amp;lt;0.01). Furthermore, pulmonary metastases were observed more often in LuCaP136 and LNCaP xenografts when PCa cells were coinjected with CAFs (57% for LuCaP136, 71% for LNCaP) as compared to coinjection with NCAFs (0% for LuCaP136, 29% for LNCaP) and BPHFs (14% for LuCaP136, 29% for LNCaP). Conclusions: Using a representative orthotopic xenograft model and patient-derived fibroblast primary cultures it could be shown that CAFs play a crucial role in local and systemic PCa progression by stimulating primary tumor growth as well as metastatic spread to the lymph nodes and lungs. However, some of the effects were cell-line specific. Future experiments will have to focus on the reproduction of these observations with the use of further sets of fibroblast primary cultures and on the unraveling of the molecular mechanisms underlying the crosstalk between PCa cells and CAFs. Citation Format: Johannes Linxweiler, Markus Hammer, Christina Körbel, Andreas Müller, Michael Stöckle, Michael D. Menger, Kerstin Junker, Matthias Saar. Cancer-associated fibroblasts stimulate tumor growth and metastatic spread in an orthotopic prostate cancer xenograft model [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 5077.

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 &amp;gt;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.

Abstract 2821: Tousled like Kinase 1 binds to and regulates the protein kinase Nek1: Its implication in prostate cancer

Abstract Introduction: Prostate Cancer (PCa) is one of the most common urological malignancies in men in the United States. Tousled Like kinases (TLKs) are involved in numerous cellular functions, including the DNA Damage Response (DDR). Through a novel proteomic approach, we have identified that NIMA kinase NEK1 strongly interacts and co-localizes with TLKs and has a role in the DDR, upstream of ATR and Chk1. In the present study we have tested Thioridazine (THD), an anti-psychotic drug and inhibitor of TLKs in inhibiting the activity of NEK1 in prostate cancer cell lines and LNCaP cell derived xenografts in NOD/SCID mice and the subsequent role of ATR-Chk1 axis following DNA damage. Material &amp; Methods: We have used a prostate cancer cell line viz. LNCaP, C4-2, C4-2b, 22Rv1, DU145 and PC3 and normal prostate cell line RWPE-1. Clonogenic activity was measured by colony formation assay. Immunoblotting was done for TLKs, NEK1, p-ATR and p-Chk1 protein in PCa cell line and tumor xenografts tissues. LNCaP xenografts in NOD/SCID mice were used in present study. We have used THD (alone) or in combination with the anti-androgen drug bicalutamide in PCa cell lines as well as in xenografts mice model. Results: Treatment with THD suppresses the outgrowth of androgen-independent (AI) colonies of LNCaP cells cultured with bicalutamide. Moreover, THD significantly inhibited the colony formation efficiency of several PCa cells in vitro (even of androgen independent lines). Intra-Peritoneal (I.P.) administration of THD or Bicalutamide long-term was not very effective in inhibiting tumor growth. In contrast, combination therapy of THD and Bicalutamide remarkably inhibited the tumor growth in LNCaP xenografts model. Further p-ATR and p-Chk1 axis was inhibited by treatment with THD and in combination with bicalutamide as evident by immunoblotting of residual tumors. In cells, following DNA damage, addition of the TLK inhibitor THD impaired ATR and Chk1 activation, indicating the existence of a TLK1&amp;gt;NEK1&amp;gt;ATR&amp;gt;Chk1 pathway. Conclusions: Our data indicated that THD repressed growth of PCa cells in vitro and in vivo, and for Androgen-dependent (LNCaP) cells, in combination with bicalutamide it was very effective at preventing outgrowth of AI cells and hence as a targeted ADT adjuvant therapy. Moreover, THD impaired DDR. Collectively, these results strongly suggest that THD might be a novel anti-tumor agent for use in prostate cancer. Citation Format: Vibha Singh, Praveen K. Jaiswal, Hari K. Koul, Xiuping Yu, Arrigo Benedetti. Tousled like Kinase 1 binds to and regulates the protein kinase Nek1: Its implication in prostate cancer [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 2821.

Abstract 838: Synergistic in vivo activity of the ATR inhibitor BAY 1895344 in combination with the targeted alpha therapy radium-223 dichloride in a preclinical tumor model mimicking bone metastatic castration-resistant prostate cancer (mCRPC)

Abstract The integrity of the genome in eukaryotic cells is secured by complex signalling pathways, known collectively as DNA damage response (DDR). Recognition of DNA damage activates DDR pathways resulting in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival, or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR pathway, such as ATR (ataxia telangiectasia and Rad3-related). The ATR kinase is activated by a broad spectrum of DNA damages, including double-strand breaks (DSBs) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in cancers characterized by increased DNA damage, deficiency in DNA damage repair or replication stress. Radium-223 dichloride (Xofigo®) is the first approved targeted alpha therapy. It is indicated for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceral metastatic disease, based on improvement of overall survival. It is a calcium-mimetics that selectively binds to hydroxyapatite targeting areas of high bone turnover such as bone metastases, thereby exhibiting strong cytotoxic effects on adjacent cells via induction of DNA DSBs. We assessed the anti-tumor efficacy of combination treatment with ATR inhibitor (ATRi) BAY 1895344 and radium-223 dichloride in an intratibially injected prostate cancer model mimicking CRPC with bone metastases. In vivo analyses addressed the optimization of the dosing schedule as well as dose-response of BAY 1895344 in radium-223 combination setting. Levels of intra-tumor DNA damage (P-H2AX) were assessed to demonstrate the proposed mode-of-action. Here, we show that combination treatment with ATRi BAY 1895344 and radium-223 exhibits synergistic anti-tumor activity in the intratibial LNCaP xenograft model of mCRPC, achieving best efficacy when BAY 1895344 is applied 24 hours after treatment with radium-223, as indicated by direct reduction of tumor burden in the bone, lower serum tumor marker (PSA), and smaller areas of tumor-induced changes in bone. With this optimized schedule, a very low dose of BAY 1895344 (3% of single-agent MTD) was effective in combination with radium-223. Our findings strongly suggest that the combination of DNA damage induction by radium-223 with DDR inhibition by ATRi BAY 1895344 creates a potential new treatment option for CRPC patients with bone metastases. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Arne Scholz, Mari Suominen, Kirstin Meyer, Eleni Lagkadinou, Dominik Mumberg. Synergistic in vivo activity of the ATR inhibitor BAY 1895344 in combination with the targeted alpha therapy radium-223 dichloride in a preclinical tumor model mimicking bone metastatic castration-resistant prostate cancer (mCRPC) [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 838.

Biphasic Alteration of Butyrylcholinesterase (BChE) During Prostate Cancer Development

Butyrylcholinesterase (BChE) is a plasma enzyme that hydrolyzes ghrelin and bioactive esters, suggesting a role in modulating metabolism. Serum BChE is reduced in cancer patients. In prostate cancer (PC), the down-regulation is associated with disease recurrence. Nonetheless, how BChE is expressed in PC and its impact on PC remain unclear. We report here the biphasic changes of BChE expression in PC. In vitro, BChE expression was decreased in more tumorigenic PC stem-like cells (PCSLCs), DU145, and PC3 cells compared to less tumorigenic non-stem PCs and LNCaP cells. On the other hand, BChE was expressed at a higher level in LNCaP cells than immortalized but non-tumorigenic prostate epithelial BPH-1 cells. In vivo, BChE expression was up-regulated in DU145 xenografts compared to LNCaP xenografts; DU145 cell-derived lung metastases displayed comparable levels of BChE as subcutaneous tumors. Furthermore, LNCaP xenografts produced in castrated mice exhibited a significant increase of BChE expression compared to xenografts generated in intact mice. In patients, BChE expression was down-regulated in PCs (n = 340) compared to prostate tissues (n = 86). In two independent PC populations MSKCC (n = 130) and TCGA Provisional (n = 490), BChE mRNA levels were reduced from World Health Organization grade group 1 (WHOGG 1) PCs to WHOGG 3 PCs, followed by a significant increase in WHOGG 5 PCs. The up-regulation was associated with a reduction in disease-free survival (P = .008). Collectively, we demonstrated for the first time a biphasic alteration of BChE, its down-regulation at early stage of PC and its up-regulation at advanced PC.

Aneustat (OMN54) has aerobic glycolysis-inhibitory activity and also immunomodulatory activity as indicated by a first-generation PDX prostate cancer model.

Aneustat (OMN54) is a multivalent, botanical anticancer candidate therapeutic. A recent Phase‐I clinical trial has indicated that it is well tolerated by patients and has immunomodulatory activity. In our study, using in vitro and in vivo prostate cancer models, we investigated Aneustat with regard to effects on (i) cancer‐generated immunosuppression based on aerobic glycolysis leading to acidification of the tumor microenvironment, and (ii) immune‐related processes such as macrophage differentiation and shifts in the intratumoral levels of host immune cells. Aneustat markedly reduced glucose consumption, lactic acid secretion, glycolysis‐related gene expressions and proliferation of human LNCaP prostate cancer cells. In addition, Aneustat induced differentiation of RAW264.7 macrophages to the M1 anticancer phenotype. Treatment of LNCaP xenografts and first‐generation patient‐derived prostate cancer tissue xenografts with Aneustat in both cases led to a marked shift in intratumoral host (mouse/patient) immune cell levels: a higher ratio of cytotoxic CD8+ T/Treg cells, higher numbers of NK cells, lower numbers of Treg cells and MDSCs, i.e. changes favoring the host anticancer immune response. Taken together, the data indicate that Aneustat has immunomodulatory activity based on inhibition of aerobic glycolysis which in patients may lead to reduction of cancer‐induced immunosuppression. Furthermore, first‐generation patient‐derived cancer tissue xenograft models may be used for screening compounds for immunomodulatory activity.

Microrna-198: A novel tumor suppressor in prostate cancer.

92 Background: microRNAs (miRNAs) are small non-coding RNA molecules which act as repressors of gene function, and have been identified as playing substantial roles in cancer as both tumor suppressors and oncogenes. miR-198 has been reported to be down-regulated in several cancers, with low expression associated with worse overall survival. In addition, miR-198 has demonstrated tumor suppressor effects by altering several hallmarks of cancer. Despite compelling evidence in other cancers, miR-198's role in prostate cancer aggression has not yet been evaluated. Methods: Experiments were conducted by overexpressing miR-198 in three prostate cancer cell lines: DU145, LNCaP and 22RV1. To examine miR-198's effect on hallmarks of cancer in vitro, we used standard protocols to assay proliferation, colony formation, cell cycle profile, migration, and invasive potential. Gene array, qPCR, western blotting, and siRNA knockdown experiments were used to establish miR-198 downstream targets. Results: Overexpression of the candidate tumor suppressor miR-198 diminished proliferation in all prostate cancer cell lines and significantly impaired colony formation in soft agar. Subsequently, miR-198 expression was also demonstrated to reduce growth and tumor formation in vivo using LNCaP xenografts. Gene arrays and in silico target prediction identified several candidate targets, none of which have been previously linked to miR-198. MIB1, an E3 ubiquitin ligase, was the only target we have since identified to be reduced with miR-198 overexpression at both the RNA and protein levels. Knockdown of MIB1 recapitulated miR-198's effects on proliferation and colony formation, and further experiments are underway to demonstrate a direct binding relationship. An additional gene array with MIB1 siRNA will be performed to highlight pathways through which MIB1/miR-198 suppress tumorigenesis. Conclusions: Our evidence supports miR-198 as an important tumor suppressor in prostate cancer, with elevated expression impairing proliferation, colony formation, and in vivo tumor formation. This investigation into miR-198 highlights a potential role as a prostate cancer biomarker, or as a potential target of therapeutics to restore miRNA activity.

SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand‐independent manner via Plexin B1. SEMA3C expression levels increase in castration‐resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide‐resistant progression. Plexin B1 sema domain‐containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post‐castration in vivo. SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy.

The 18-membered macrocycle H2 macropa was investigated for 225 Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225 Ac (26 kBq) in 5 min at RT. [225 Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa-NCS, was conjugated to trastuzumab as well as to the prostate-specific membrane antigen-targeting compound RPS-070. Both constructs rapidly radiolabeled 225 Ac in just minutes at RT, and macropa-Tmab retained >99 % of its 225 Ac in human serum after 7 days. In LNCaP xenograft mice, 225 Ac-macropa-RPS-070 was selectively targeted to tumors and did not release free 225 Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225 Ac chelation that will facilitate the clinical development of 225 Ac TAT for the treatment of soft-tissue metastases.

An Eighteen‐Membered Macrocyclic Ligand for Actinium‐225 Targeted Alpha Therapy

AbstractThe 18‐membered macrocycle H2macropa was investigated for 225Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225Ac (26 kBq) in 5 min at RT. [225Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa‐NCS, was conjugated to trastuzumab as well as to the prostate‐specific membrane antigen‐targeting compound RPS‐070. Both constructs rapidly radiolabeled 225Ac in just minutes at RT, and macropa‐Tmab retained &gt;99 % of its 225Ac in human serum after 7 days. In LNCaP xenograft mice, 225Ac‐macropa‐RPS‐070 was selectively targeted to tumors and did not release free 225Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225Ac chelation that will facilitate the clinical development of 225Ac TAT for the treatment of soft‐tissue metastases.

Assessment of PSMA targeting ligands bearing novel chelates with application to theranostics: Stability and complexation kinetics of 68Ga3 +, 111In3 +, 177Lu3 + and 225Ac3 +

IntroductionRecent successes in the treatment of metastatic castration-resistant prostate cancer (mCRPCa) by systemic endoradiotherapy has sparked renewed interest in developing small molecule ligands targeting prostate-specific membrane antigen (PSMA) and chelators capable of stable complexation of metal radionuclides for imaging and therapy. As the size and coordination number of metals for imaging, such as 68Ga3+, and for targeted therapy, such as 177Lu3+ and 225Ac3+, are substantially different, they may show a preference for macrocycles of different denticity. We have prepared three simple conjugates that target PSMA and form radiometal complexes through coordination by either octa-, deca-, or dodecadentate tetraazacyclododecane chelators. The complex formation and metal ion selectivity of these constructs were determined at two relevant temperatures, complex stability was examined in vitro, and tumor targeting was demonstrated in preclinical PCa models with a view towards identifying a candidate with potential value as a theranostic agent for the imaging and therapy of mCRPCa. MethodsThree bifunctional chelates with high denticity, including the octadentate chelate DOTA, the decadentate 3p-C-DEPA and a novel dodecadentate analogue of DEPA, were synthesized and conjugated to a glutamate-urea-lysine (EuK) pharmacophore (EuK-DOTA, EuK-107 and EuK-106, respectively) to enable targeting of PSMA. The metal ion selectivity for each construct was determined by incubation at 25 °C and 95 °C with the trivalent radiometals 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+. PSMA binding affinity was determined by competitive binding using LNCaP cells, while in vivo tumor targeting of the 68Ga-labeled constructs was examined by positron emission tomography (PET) in LNCaP xenograft tumor-bearing mice. ResultsPMSA affinities (IC50 values) were 13.3 ± 0.9 nM for EuK-DOTA, 18.0 ± 3.7 nM for EuK-107 and 42.6 ± 6.6 nM for EuK-106. EuK-107 and EuK-DOTA proved to rapidly and near quantitatively complex 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+ at 95 °C, with EuK-107 also rapidly complexing 111In3+ and 177Lu3+ at 25 °C. The inability of EuK-106 to chelate 177Lu3+ and 225Ac3+ suggests that size of the cavity of the macrocylic ring may be more critical than the number of donor groups for the chelation of larger radiometals. In vivo, 68Ga-EuK-107 proved to have similar uptake to 68Ga-DKFZ-PSMA-617, a theranostic ligand currently in clinical evaluation, in a PSMA positive xenograft tumor model. ConclusionsThe broad metal ion selectivity, good in vitro affinity for PSMA and good in vivo tumor targeting suggest that EuK-107, with the 3p-C-DEPA chelator, merits further evaluation as a theranostics construct in prostate cancer.

Overexpression of MUC1 and Genomic Alterations in Its Network Associate with Prostate Cancer Progression

We investigate the association of MUC1 with castration-resistant prostate cancer (CRPC), bone metastasis, and PC recurrence. MUC1 expression was studied in patient-derived bone metastasis and CRPCs produced by prostate-specific PTEN−/− mice and LNCaP xenografts. Elevations in MUC1 expression occur in CRPC. Among nine patients with hormone-naïve bone metastasis, eight express MUC1 in 61% to 100% of PC cells. Utilizing cBioPortal PC genomic data, we organized a training (n=300), testing (n=185), and validation (n=194) cohort. Using the Cox model, a nine-gene signature was derived, including eight genes from a MUC1-related network (APC, CTNNB1/β-catenin, GALNT10, GRB2, LYN, SIGLEC1, SOS1, and ZAP70) and FAM84B. Genomic alterations in these genes reduce disease-free survival (DFS) in the training (P=.00161), testing (P=.00699), entire (training+testing, P=5.557e-5), and a validation cohort (P=3.326e-5). The signature independently predicts PC recurrence [hazard ratio (HR)=1.731; 95% confidence interval (CI): 1.104-2.712; P=.0167] after adjusting for known clinical factors and stratifies patients with high risk of PC recurrence using the median (HR 2.072; 95% CI: 1.245-3.450, P=.0051) and quartile 3 (HR 3.707, 95% CI: 1.949-7.052, P=6.51e-5) scores. Several novel β-catenin mutants are identified in PCs leading to a rapid onset of death and recurrence. Genomic alterations in APC and CTNNB1/β-catenin reduce DFS in two independent PC cohorts (n=485, P=.0369; n=84, P=.0437). The nine-gene signature also associates with reductions in overall survival (P=.0458) and DFS (P=.0163) in melanoma patients (n=367). MUC1 upregulation is associated with CRPC and bone metastasis. A nine-gene signature derived from a MUC1 network predicts PC recurrence.