Prostate Cancer Cell Lines VCaP Research Articles

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Comparative Proteomic and Transcriptomic Analysis of the Impact of Androgen Stimulation and Darolutamide Inhibition.

Several inhibitors of androgen receptor (AR) function are approved for prostate cancer treatment, and their impact on gene transcription has been described. However, the ensuing effects at the protein level are far less well understood. We focused on the AR signaling inhibitor darolutamide and confirmed its strong AR binding and antagonistic activity using the high throughput cellular thermal shift assay (CETSA HT). Then, we generated comprehensive, quantitative proteomic data from the androgen-sensitive prostate cancer cell line VCaP and compared them to transcriptomic data. Following treatment with the synthetic androgen R1881 and darolutamide, global mass spectrometry-based proteomics and label-free quantification were performed. We found a generally good agreement between proteomic and transcriptomic data upon androgen stimulation and darolutamide inhibition. Similar effects were found both for the detected expressed genes and their protein products as well as for the corresponding biological programs. However, in a few instances there was a discrepancy in the magnitude of changes induced on gene expression levels compared to the corresponding protein levels, indicating post-transcriptional regulation of protein abundance. Chromatin immunoprecipitation DNA sequencing (ChIP-seq) and Hi-C chromatin immunoprecipitation (HiChIP) revealed the presence of androgen-activated AR-binding regions and long-distance AR-mediated loops at these genes.

Inhibition of N-Acetyltransferase 10 Suppresses the Progression of Prostate Cancer through Regulation of DNA Replication.

Cancer suppression through the inhibition of N-acetyltransferase 10 (NAT10) by its specific inhibitor Remodelin has been demonstrated in a variety of human cancers. Here, we report the inhibitory effects of Remodelin on prostate cancer (PCa) cells and the possible associated mechanisms. The prostate cancer cell lines VCaP, LNCaP, PC3, and DU145 were used. The in vitro proliferation, migration, and invasion of cells were measured by a cell proliferation assay, colony formation, wound healing, and Transwell assays, respectively. In vivo tumor growth was analyzed by transplantation into nude mice. The inhibition of NAT10 by Remodelin not only suppressed growth, migration, and invasion in vitro, but also the in vivo cancer growth of prostate cancer cells. The involvement of NAT10 in DNA replication was assessed by EdU labeling, DNA spreading, iPOND, and ChIP-PCR assays. The inhibition of NAT10 by Remodelin slowed DNA replication. NAT10 was detected in the prereplication complex, and it could also bind to DNA replication origins. Furthermore, the interaction between NAT10 and CDC6 was analyzed by Co-IP. The altered expression of NAT10 was measured by immunofluorescence staining and Western blotting. Remodelin markedly reduced the levels of CDC6 and AR. The expression of NAT10 could be altered under either castration or noncastration conditions, and Remodelin still suppressed the growth of in vitro-induced castration-resistant prostate cancers. The analysis of a TCGA database revealed that the overexpression of NAT10, CDC6, and MCM7 in prostate cancers were correlated with the Gleason score and node metastasis. Our data demonstrated that Remodelin, an inhibitor of NAT10, effectively inhibits the growth of prostate cancer cells under either no castration or castration conditions, likely by impairing DNA replication.

Abstract 1008: Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model

Abstract Androgen receptor (AR) signaling is the major driver in prostate cancer. Darolutamide is a high-affinity AR inhibitor recently approved for non-metastatic castration-resistant prostate cancer. The impact of the AR on gene transcription has been extensively examined but the downstream effects on protein levels are still poorly understood. Here we generated for the first time comprehensive quantitative proteomic and transcriptomic data from the androgen-sensitive, prostate cancer cell line VCaP treated with the androgen R1881 and darolutamide. For the proteomic analysis, a comprehensive and quantitative label-free protein expression profiling was performed using nanoscale liquid chromatography coupled to tandem mass spectrometry. For the transcriptomic analysis, sequencing was performed via single-end, 50 base-pair reads with an average depth of 21 million reads per sample. FASTQ reads were mapped with STAR aligner to the human genome GRCh38 and quantified with RSEM. Further analysis was performed in R statistical software. We investigated the expression levels of 12900 protein-coding genes and 8721 translated proteins. Androgen treatment resulted in increased expression of known androgen response genes, as well as of genes from the mTORC1 pathway, cell cycle and other pathways previously shown to be affected by androgens. This was paralleled by upregulation of the corresponding protein levels in the majority of cases. Importantly, a detailed comparative analysis of transcriptomic and proteomic data revealed a few notable differences in regulation. In androgen-treated prostate cancer cells, there was a strong increase of FOLH1, AMACR and FKBP5 transcript levels (10 to 40-fold) but only a moderate increase of the corresponding protein levels (2 to 6-fold). Additional darolutamide treatment led to strong down-regulation of androgen-induced gene expression and of the corresponding protein levels. Here also there were a few instances where protein and transcript levels were differentially regulated. The discrepancy in transcript and protein levels seen after androgen and anti-androgen treatment suggests differential post-transcriptional regulation of protein abundance to occur in some instances. In conclusion, we found that darolutamide strongly reversed androgen-induced activation of gene expression programs related to cell proliferation and survival, both at the transcript and protein levels. We furthermore identified for the first time androgen-activated genes whose function is additionally regulated at the post-transcription level. Citation Format: Ekaterina Nevedomskaya, Simon J. Baumgart, Tatsuo Sugawara, Ralf Lesche, Dominik Mumberg, Bernard Haendler. Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer 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 1008.

Abstract 1753: Targeting cancer with selective cbp/p300 bromodomain inhibitors

Abstract Background: The Bromodomain (BRD) and Extra-Terminal domain (BET) family of proteins are key regulators of epigenetic control. Although pan BET Inhibitors show good clinical activity, progressive disease was seen after several months of treatment in clinical responders, likely due to secondary resistance mechanisms. The plausible resistance mechanisms are increased expression of TCF7L2, c-Myc, Survivin and PIM1. Cyclic AMP response element binding protein (CREB)-binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or p300) are two closely related histone acetyl transferases with oncogenic roles in a variety of cancers. They are known to be co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Studies have indicated that p300 is also upregulated in SPOP-mutated prostate cancer. Thus, targeting CBP/p300 represents an attractive approach for developing novel therapies. Methods and Results: Multiple potent and selective CBP/p300 bromodomain inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The compounds were optimized towards attaining good physicochemical properties and DMPK profile. The anti-proliferative activity of the lead compounds was studied across multiple tumor types in a 3-day assay. The lead compounds potently inhibited viability of a wide range of hematological and solid tumor cell lines including prostate cancer cell lines VCaP and 22Rv1. In H929 cell line the lead compounds showed dose-dependent inhibition of cMYC and increase in cPARP. In a single dose PK-PD study in MV4-11 xenograft model, the compounds showed modulation of cMYC and Survivin. Conclusions: In summary, our studies demonstrate that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in-vitro. Profiling of efficacy in xenograft models, and further toxicological evaluation are in progress. Citation Format: Mahaboobi Jaleel, Ramesh S. Senaiar, Chandrasekhar Abbineni, Girish A. Renukappa, Subhendu Mukherjee, Sivapriya Marappan, DS Samiulla, AB Aravind, Naveen R. Kumar, Venkata Siva N. Reddy, Asha Babu, Akhila P. Srinivas, Prasad Yadlapalli, Suraj Tgore, Raghavendra NR, Chandranath D. Naik, Sanjeev Giri, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, laura Ravanti, Mikko Myllymäki, Gerd Wohlfahrt, Elina Mattila, Stefan Karlsson, Mari Björkman, Reetta Riikonen, Tarja Ikonen, Laura Leimu, Chira Mälmström, Timo Korjamo, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. Targeting cancer with selective cbp/p300 bromodomain inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1753.

MiR-133a-5p inhibits androgen receptor (AR)-induced proliferation in prostate cancer cells via targeting FUsed in Sarcoma (FUS) and AR

ABSTRACTAndrogens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly employed to treat advanced prostate cancer. Previously, FUsed in Sarcoma (FUS) was identified as an AR-interacting protein that enhances AR transcriptional activity. In the present study, we attempted to identify miRNAs that might target both FUS and AR to inhibit FUS and AR expression. Based on TCGA data and the online tools UALCAN, Kaplan Meier-plotter (KMplot), LncTar and miRWalk prediction, miR-133a-5p was selected. MiR-133a-5p expression was significantly downregulated in prostate cancer, and low miR-133a-5p expression was correlated with low survival probability. As predicted by LncTar and miRWalk, miR-133a-5p could bind to the 3′UTR of FUS and AR to inhibit their expression. MiR-133a-5p overexpression significantly suppressed the cell viability of the AR-positive prostate cancer cell lines VCaP and LNCaP, inhibited the expression of FUS, AR, as well as AR downstream targets IGF1R and EGFR. More importantly, miR-133a inhibition increased cancer cell proliferation as well as the expression of AR and AR downstream factors, while FUS knockdown exerted an opposite effect; the effect of miR-133a on cancer cell proliferation and AR could be significantly reversed by FUS knockdown. Moreover, IGF1R and EGFR knockdown reversed the effect of the miR-133a-5p inhibition. In summary, miR-133a-5p inhibits AR-positive prostate cancer cell proliferation by targeting FUS/AR, thus improving the resistance of prostate cancer to androgen ablation therapies, which requires further in vivo validation. We provided a novel miRNA regulation mechanism for proliferation regulation in AR-positive prostate cancer cells.

Abstract 2772: Recurrent alterations of the TenascinC in highly aggressive neuroendocrine sub-type of prostate cancer

Abstract Introduction: Early detection and prognosis of prostate cancer (CaP) is challenging due to its wide spectrum of biological features. Multi-analytic prognostic marker panels have increased sensitivity than single analytes among the biopsy tests and require advanced bioinformatics platform. We aimed to develop a panel of serum biomarkers using multi-omics approach and further characterized their involvement in prostate cancer progression. In a collaborative study between CPDR, Department of Surgery, USU and Berg Health, serum samples (N=385) were examined by multi-omics (proteomics, lipidomics and metabolomics) and Tenascin C (TNC) was identified as one of the promising markers for disease progression in combination with three other analytes, Apolipoprotein AIV, 1-Methyladenosine and a phosphatidic acid (PA 18:0-22:0). The combination of two clinical features, pathological measurement of T-Stage and Gleason score, along with the four molecular analytes further increased the AUC to 0.89 with a NPV of 0.96 and an odds ratio of 12.4. TNC, an extracellular matrix protein, is poorly studied in prostate cancer, though it is expressed in several cancer tissues such as the breast, lung, colon, and the gastrointestinal tract. Methods: Publically available prostate cancer databases (cBioportal Version 1.17.1, http://www.cbioportal.org/index.do) were queried for TNC, and known driver oncogenes in prostate cancer, ERG, AR and MYC. Results: Publically available prostate cancer databases (cBioPortal) demonstrated alterations (either amplifications or mutations) in TNC in 10 out of 16 datasets. Query of TNC along with the driver oncogenes of prostate tumorigenesis, including ERG, MYC and AR, in the aggressive neuroendocrine prostate cancer (NEPC) tumor whole genome sequencing datasets (N=77) demonstrating significant alterations (predominantly amplifications) in 74% of patient samples. CaP genomic levels TNC (30%) was significantly co-amplified (P < 0.001) with ERG (27%), AR (56%) and MYC (53%). TNC protein expression was detected in all examined prostate cancer cell lines VCaP, LNCaP, PC3 and DU145. Conclusion: Genomic alterations of TNC was thus associated with major oncogenic drivers of CaP, such as ERG, AR and MYC in NEPC genomic datasets. Multi-analyte serum biomarkers offers new opportunities with potential impact on primary treatment and surveillance strategies. Functional involvement of the analytes in disease progression to address their mechanistic link with major CaP oncogenic pathways will be further investigated. Citation Format: Prachi Mishra, Michael Kebish, Jennifer Cullen, Amina Ali, Alagasamy Srinivasan, Inger Rosner, David McLeod, Leonardo Rodrigues, Viatcheslav Akmaev, Rangaprasad Sarangarajan, Shiv Srivastava, Niven Narain, Albert Dobi. Recurrent alterations of the TenascinC in highly aggressive neuroendocrine sub-type of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2772.

Abstract 3746: Targeting adrenal androgen to treat prostate cancer

Abstract Background: Adrenal androgens are the most abundant androgens in humans, with dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) being the major adrenal androgens in circulation. Significantly, adrenal androgens may serve as substrates for intratumoral (intracrine) production of testosterone (T) and/or dihydrotestosterone (DHT), the ligands for androgen receptor (AR). However, adrenal androgens also have functions that are independent of AR. The present study addressed the capability of prostate cancer tissue to use adrenal androgens for T/DHT production, delineated signaling pathways activated by adrenal androgens, and determined whether adrenal androgens sustained tumor growth after androgen-deprivation therapy (ADT). Experimental Procedures: Metabolism of adrenal androgen was evaluated ex vivo using fresh clinical prostate tissue specimens. The production of DHT or DHEA was determined using ELISA. Human prostate cancer cells lines were used for in vitro experiments. The effects of adrenal androgens on tumor growth were evaluated in vivo using xenografts of the human prostate cancer cell line VCaP in castrated or intact mice. Results: 1) Human prostate tissue was able to produce DHT using DHEA-S at a physiologic concentration (3.5 μM). The production of DHT from DHEA-S was absolutely dependent on the activity of steroid sulfatase (STS). DHEA at a physiologic concentration (10 nM) was not a suitable substrate for DHT production, but was used by prostate tissue when available at a supra-physiologic concentration (3.5 μM) to produce DHT. 2) DHEA-S was able to stimulate growth of prostate cancer cells in vitro. The growth stimulatory effect of DHEA-S was mediated by AR-dependent and AR-independent signaling pathways. A subset of the genes modulated specifically by DHEA-S were distinctive from the T-modulated genes. 3) DHEA sustained tumor growth after castration. DHEA-stimulated growth was attributed to activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway. Conclusions: Prostate tissue can use adrenal androgens for DHT production. Adrenal androgens may stimulate prostate cancer growth using AR-dependent pathways that are mediated via intratumoral T/DHT production, or using AR-independent signaling pathways such as the IGF1R signaling pathway. Identification of targets and therapeutics to block the adrenal androgen-stimulated pathways may help develop novel strategies to treat prostate cancer. Citation Format: Yue Wu, Jianmin Wang, Li Tang, Elena Pop, John J. Stocking, Kristopher M. Attwood, Gary J. Smith. Targeting adrenal androgen to treat 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 3746.

Abstract B170: Novel immunodeficient rat models offer a unique platform for drug efficacy studies in human tumor xenografts

Abstract Immunodeficient mouse models seek to recapitulate the clinical features of advanced human cancers. However, drug efficacy testing and downstream analysis such as pharmacokinetic (PK) /pharmacodynamic (PD)-based studies are limited because of variability in tumor growth and kinetics, limited tumor growth potential, requiring the enrollment of larger numbers of mice in each treatment group to achieve required cohort sizes. Considering the distinctive advantages of rats in terms of their larger size, and ease of sampling at different time points for drug efficacy and clinical testing, we have developed unique immunodeficient rat models: the Rag2 knockout SDRTM rat (absence of mature B and T cells) and the Rag2/Il2rg double knockout SRGTM rat (absence of mature B and T cells, and lower NK cells) on a Sprague- Dawley background. Our group has previously demonstrated higher engraftment, better tumor kinetics, and increased tumor volume of xenografts in these rat models compared to available immunodeficient mouse strains for various cancer xenograft models. The purpose of the present study is to demonstrate the feasibility of drug efficacy studies using targeted molecular agents directed against cancer-associated drivers in our newly developed SDR and SRG rat models. We tested the efficacies of a combination treatment of an AKT inhibitor (MK2206) and MEK inhibitor (AZD6244), and a small-molecule activator of PP2A (SMAP) (DT-061) in a SDR rat xenograft model of human KRAS-mutant non-small cell lung cancer. 1 x 106 H358 cells were transplanted subcutaneously on the left hind flank in SDR rats. When tumor size reached 100-150 mm3, the rats were randomized to either control or treatment groups. The treatment groups received either a combination of AZD6244 with MK2206, or DT-061, and the vehicle-only control received n, n-dimethylacetamide + Solutol®/Kolliphor® HS 15, twice daily for 30 days by oral gavage. Both the kinase-inhibitors combination and SMAP treatment inhibited the growth of lung tumor xenograft in the SDR rats as shown by decreased mean tumor volume and fold changes in tumor volume compared to the control group. The combination treatment dose was just as effective at half the dose reported in previous mouse studies, demonstrating that lower dosing is sufficient for efficacy testing in rats. Importantly, no mortality, behavioral abnormalities, or changes in body weight were observed during the study, indicating a favorable tolerability profile in this model. We have also demonstrated that a human prostate cancer cell line VCaP, which has very poor engraftment and growth profiles in existing mouse models, grows well in our SRG rats. The SRG rat is now being used to study the chemotherapeutic efficacy of the benchmark androgen receptor inhibitor enzalutamide, in this VCaP tumor xenograft model. In addition, weekly blood sampling to measure PSA levels in the serum is being performed in the VCaP tumor xenograft efficacy study to demonstrate the flexibility of frequent sampling in the rat model to evaluate correlations between tumor growth and serum PSA levels. Citation Format: Kamesh Ravi, Fallon Noto, Christopher McClain, Angela Arey, Goutham Narla, Jack Crawford, Tseten Yeshi. Novel immunodeficient rat models offer a unique platform for drug efficacy studies in human tumor xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B170.

Abstract 20: Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer

Abstract Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. A large body of preclinical evidence suggests that IGF signaling plays a key role in cancer by driving therapy resistance, due to cross-talk with other signaling networks such as androgen receptor signaling. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[1]), to enhance the anti-tumor activity of enzalutamide in prostate cancer cell lines and in a patient-derived prostate cancer xenograft model. Methods: Effects of enzalutamide, xentuzumab and combinations thereof on in vitro proliferation, survival, cell cycle and signaling were evaluated using the prostate cancer cell lines VCaP, DuCaP, MDA PCa 2b, and LNCaP. The in vivo efficacy of enzalutamide, alone and in combination with xentuzumab was investigated using LuCaP 96CR, a patient-derived xenograft model of castration-resistant prostate cancer. Tumors were implanted s.c. into castrate SCID mice. When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) enzalutamide (50 mg/kg QD po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW ip) in combination with enzalutamide. Results: Cell viability was more effectively reduced by the combination of enzalutamide and xentuzumab than either drug alone in three of four cell lines expressing the IGF-1R and the androgen receptor (AR). In VCaP cells, prolonged inhibition of IGF pathway signaling and enhanced blockade of proliferation as well as induction of apoptosis was observed after combination treatment. In vivo, enzalutamide monotherapy did not show significant antitumor efficacy in the LuCaP 96CR model, however, combined treatment with xentuzumab significantly inhibited progression of LuCaP 96CR tumor growth (p<0.001 vs. enzalutamide alone). Reduced serum PSA levels were observed after enzalutamide and combination treatment. Enzalutamide plus xentuzumab inhibited tumor growth at tolerated doses and resulted in significant improvements in survival. Conclusions: These studies demonstrated that addition of the IGF-1/-2 neutralizing antibody xentuzumab to enzalutamide results in improved anti-neoplastic activity in a subset of prostate cancer cell lines in vitro, and to re-sensitization to enzalutamide in a patient-derived xenograft model of CRPC. Reference: [1] Friedbichler K et al. (2014). Mol Cancer Ther 13(2):399-409. Citation Format: Ulrike Weyer-Czernilofsky, Marco H. Hofmann, Paul J. Adam, Flavio Solca, Katrin Friedbichler, Norbert Kraut, Eva Corey, Thomas Bogenrieder. Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 20. doi:10.1158/1538-7445.AM2017-20

Oncolytic alphavirus SFV-VA7 efficiently eradicates subcutaneous and orthotopic human prostate tumours in mice

Background:Despite recent therapeutic and diagnostic advances, prostate cancer remains the second leading cause of cancer-related deaths among men in the Western world. Oncolytic viruses that replicate selectively in tumour cells represent a novel treatment candidate for these malignancies.Methods:We analysed infectivity of avirulent Semliki Firest virus SFV-VA7 in human prostate cancer cell lines VCaP, LNCaP and 22Rv1 and in nonmalignant prostate epithelial cell line RWPE-1. Therapeutic potency of SFV-VA7 was evaluated in subcutaneous and orthotopic mouse LNCaP xenograft models.Results:SFV-VA7 infected and killed the tested human prostate cancer cell lines irrespective of their hormone response status, while the nonmalignant prostate epithelial cell line RWPE-1 proved highly virus resistant. Notably, a single peritoneal dose of SFV-VA7 was sufficient to eradicate all subcutaneous and orthotopic LNCaP tumours.Conclusions:Our results indicate that SFV-VA7 is a novel, promising therapeutic virus against prostate cancer warranting further testing in early clinical trials.

Abstract A62: Tenascin C as an effector of prostate cancer derived bone metastasis

Abstract The purpose of this study is to characterize the microenvironment changes in prostate cancer bone metastasis in the context of a reactive stroma response, and to evaluate how these changes affect metastatic colonization and proliferation. The stroma plays an important role in the maintenance of tissue homeostasis. In particular, stroma associated with secretory epithelium, like prostate acini, must be able to initiate an efficient wound repair response in the event of a breach in the epithelial layer in order to prevent infection or further damage. This reactive stroma response is characterized by the accumulation of myofibroblasts, also known as carcinoma associated fibroblasts (CAFs) followed by remodeling of extracellular matrix at the site of injury. The reactive stroma response initiates early in prostate cancer, co-evolves with disease progression, and is predictive of recurrence. One way reactive stroma remodels the microenvironment is by deposition of tenascin-C. Tenascin-C is an extracellular matrix protein that is expressed during development and is critical for neuronal patterning and osteogenesis. In contrast, expression of tenascin C in adult tissues is limited and is restricted to regions of wound repair, tissue remodeling and pathological conditions, such as cancer. To assess whether the bone exhibits a reactive tissue phenotype in the context of metastasis, human prostate cancer metastasis tissue arrays were evaluated using immunohistochemistry and spectral deconvolution. This work identified a tenascin-C expression pattern at trabeculae-associated metastatic sites, suggesting the evolution of a reactive endosteum. In order to evaluate the mechanisms involved, we developed an in vitro 3D osteogenic organoid, using human mesenchymal stem cells induced to osteoblastic differentiation, which exhibits a reactive endosteum phenotype. Co-culture with the metastatic prostate cancer cell line VCaP showed preferential binding at sites high in tenascin C deposition. Also, metastatic cells were capable of adherence to purified tenascin C in vitro, forming 3D colonies. We have identified α9β1 integrin as a mediator of prostate cancer cell adhesion to tenascin C-rich surfaces. Preliminary data has also identified signaling through WNK1 and STAT6 as additional candidate mechanisms that mediate tenascin-C induced biology in prostate cancer cells that are metastatic to bone surfaces. These studies characterize a reactive endosteum phenotype at sites of metastatic prostate cancer foci and suggest that elevated tenascin-C at these sites mediates adhesion and other biological properties of cancer cells. This study will help provide data from which to develop novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, Kenneth Pienta, David R. Rowley. Tenascin C as an effector of prostate cancer derived bone metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A62.

Abstract 2268: Tenascin C as an effector of prostate cancer derived bone metastasis

Abstract The stroma plays a key role in maintenance of tissue homeostasis. A reactive stroma evolves early in the progression of prostate cancer. One way reactive stroma remodels the microenvironment is by deposition of tenascin-C. Tenascin-C is an extracellular matrix protein that is expressed during development and is critical for neuronal patterning and osteogenesis. In contrast, expression of tenascin C in adult tissues is limited and is restricted to regions of wound repair, tissue remodeling and pathological conditions, such as cancer. To assess whether the bone exhibits a reactive tissue phenotype in the context of metastasis, human prostate cancer metastasis tissue arrays were evaluated using immunohistochemistry and spectral deconvolution. This work identified a tenascin-C expression pattern at trabeculae-associated metastatic sites, suggesting the evolution of a reactive endosteum. In order to evaluate the mechanisms involved, we developed an in vitro 3D osteogenic organoid, using human mesenchymal stem cells induced to osteoblastic differentiation, which exhibits a reactive endosteum phenotype. Co-culture with the metastatic prostate cancer cell line VCaP showed preferential binding at sites high in tenascin C deposition. Also, metastatic cells were capable of adherence to purified tenascin C in vitro, forming 3D colonies. We have identified α9β1 integrin as a mediator of prostate cancer cell adhesion to tenascin C-rich surfaces. Preliminary data has also identified signaling through WNK1 and STAT6 as additional candidate mechanisms that mediate tenascin-C induced biology in prostate cancer cells that are metastatic to bone surfaces. These studies characterize a reactive endosteum phenotype at sites of metastatic prostate cancer foci and suggest that elevated tenascin-C at these sites mediates the adhesive and other biological properties of cancer cells. This study will help provide data from which to develop novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, David R. Rowley, Kenneth J. Pienta. Tenascin C as an effector of prostate cancer derived bone metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2268. doi:10.1158/1538-7445.AM2015-2268

Identification of TDRD1 as a direct target gene of ERG in primary prostate cancer

Molecular classification of ERG-rearranged prostate cancer clarifies the role of TMPRSS2-ERG in the development and progression of prostate cancer. The objective of our study was to identify direct ERG target genes in ERG-rearranged prostate cancer. Two independent cohorts of primary prostate cancer (Cohort A, n=48; Cohort B, n=31), a cohort of late-stage prostate cancer (n=51) and expression array data of a cohort of primary prostate tumors from a different institute (n=128) were analyzed for expression of genes that were coexpressed with ERG overexpression. By genome-wide expression analysis and Q-RT-PCR it was shown that the gene Tudor domain containing 1 (TDRD1) was by far the strongest correlated gene with ERG overexpression in both Cohort A and B. Expression array analysis of the patient cohort from a different institute showed a large overlap in genes that were positively correlated with ERG overexpression, including TDRD1. In late-stage prostate cancer, TDRD1 was also coexpressed with ERG overexpression, although a proportion of ERG-negative late-stage samples expressed TDRD1. TDRD1 expression was not associated with ETV1 overexpression. In the prostate cancer cell line VCaP, downregulation of ERG by shRNA lead to a lower expression level of TDRD1 and resulted in a decreased activity of the TDRD1 promoter. By mutation analysis we identified a functional ERG binding site in the TDRD1 promoter. Our findings show TDRD1 as the first identified upregulated direct ERG target gene that is strongly associated with ERG overexpression in primary prostate cancer.

Abstract 3526: Subcutaneous administration of PSMA/CD3-bispecific BiTE antibody MT112/BAY 2010112 leads to complete remission of human prostate cancer xenografts in mice

Abstract Blinatumomab, a CD19/CD3-bispecific BiTE antibody has shown high response rates and durable remissions in patients with relapsed or refractory acute lymphocytic leukemia and non-Hodgkin's lymphoma. For treatment of patients with prostate cancer (PCa), we have developed a novel BiTE antibody, MT112/BAY2010112, that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. MT112/BAY 2010112 binds PSMA and CD3 of human and macaque origin allowing for assessment of safety, pharmacodynamics and pharmacokinetics in a relevant animal species. PSMA expressing human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC3-PSMA and LNCaP were lysed by freshly isolated, unstimulated human T cells redirected by MT112/BAY2010112 at EC50 values ranging between 0.1 and 4 ng/ml (1.8-72 pM). Efficacy of lysis correlated with levels of surface expressed PSMA. No lysis was observed in PSMA-negative PCa cell lines PC3 and DU145, showing the target antigen-specific activity of MT112/BAY2010112. The relevance of cynomolgus monkeys for further nonclinical safety evaluation of MT112/BAY 2010112 was demonstrated by in vitro side-by-side comparison of the BiTE antibody's pharmacological characteristics in human and cynomolgus monkey assay systems. For studying the activity of MT112/BAY 2010112 after subcutaneous (s.c.) administration, human 22Rv1 PCa xenografts were grown s.c. in mice to sizes of >200 mm3 in the absence of human T cells. Three days prior to treatment with BiTE, ex-vivo expanded human T cells were intraperitoneally injected. Animals were treated s.c. at 2.5 mg/kg/d or intravenously (i.v.) at 0.5 mg/kg/d for 28 consecutive days. S.c. administration of MT112/BAY 2010112 resulted in a similar drug exposure, rapid shrinkage and complete remission of established PCa xenografts as seen upon i.v. administration of the BiTE antibody. Treatment with human T cells or vehicle alone had no effect on tumor growth. Compared to i.v. administration, the relative bioavailability of the PSMA BiTE in serum after s.c. injection in mice was approximately 18%. Initial results will be presented showing that 14C-labeled MT112/BAY 2010112 accumulates in s.c. implanted LNCaP PCa tumors of mice after tail vein injection. Based on these preclinical in vivo pharmacology results, treatment of PCa patients with s.c. administered MT112/BAY2010112 appears to be justified. 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 3526. doi:1538-7445.AM2012-3526

The molecular evolution of prostate cancer cell line exosomes with passage number.

e21071 Background: Exosomes are endosome-derived vesicles secreted by multiple cell types, including tumor cells. The vesicles are formed intracellularly by invagination followed by fusion with the multivesicular body (MVB). The MVB merges with the plasma membrane, resulting in the exocytosis of exosomes which have membrane protein composition similar to their cell of origin. These circulating vesicles have been shown to participate in cellular communication by transporting mRNAs, microRNAs (miRs) and proteins to target cells, eliciting biological responses. This study examined the dynamics of exosome biosignatures from prostate cancer (PCa) cell lines. Methods: Exosome-associated RNA and surface membrane protein profiles from the prostate cancer cell line VCaP were analyzed by flow sorting (FACS) and a multiplexed immunoassay. The surface membrane protein composition and RNA content of VCaP exosomes were measured through twenty cell line passages. Results: The surface membrane protein composition of VCaP...

Abstract 3246: Important differences between exosome biosignatures from prostate cancer patient plasma samples and prostate cancer cell lines

Abstract Exosomes are endosome-derived vesicles secreted by many cell types, including tumor cells. The vesicles are formed intracellularly by invagination followed by fusion with the multivesicular body (MVB). The MVB ultimately merges with the plasma membrane, leading to exocytosis of the exosomes, which consequently have a similar membrane protein composition as their cell of origin; this provides the exosomes with unique biosignatures. Furthermore, these circulating exosomes participate in cellular communication by transporting mRNAs, microRNAs and proteins to target cells where they can elicit biological responses. This study compared the biosignatures identified from prostate cancer (PCa) cell line-derived exosomes to those found in exosomes isolated from the plasma of PCa patient samples. To develop an exosome-specific protein and RNA signature for PCa, we studied RNA and surface membrane protein profiles by flow cytometry (FACS) of exosomes derived from four prostate cancer cell lines, VCaP, 22Rv1, LNCaP and DU145. The exosome biosignatures identified from the cell lines were then measured in plasma exosomes isolated from PCa patients. We found that there was variability between the four cell line exosome-specific mRNA expression levels and exosome surface protein content. The mRNA exosome biosignatures identified in the four cell lines were not found in the exosomes from plasma samples of patients with PCa. Using a combination of antibodies for B7H3, PSMA and CD63 by FACS, we identified a protein signature from all prostate cancer cell line exosome populations that defined a specific subpopulation of exosomes containing all 3 proteins on their surface. Interestingly, this same exosome subpopulation was not found in exosomes derived from patients with PCa. Additionally, exosome-specific mRNA expression of two mRNA transcripts often found to be overexpressed in PCa, STEAP1 and SPINK1, were consistently identified in exosomes derived from prostate cancer cell lines VCaP and 22Rv1, but were only found in the plasma derived exosomes from one out of eight patients with PCa. Our results show that prostate cancer cell lines are not appropriate models for the development of exosome-specific disease profiles in prostate cancer; this will be investigated in additional cell lines. We find that patient samples are essential to identify disease-specific exosome biosignatures. These findings support the development of a versatile diagnostic platform based on exosome-specific biosignatures found in the plasma of patient samples for prostate and other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3246.

Role of the TMPRSS2-ERG gene fusion in prostate cancer: Tomlins SA, Laxman B, Varambally S, Cao X, Yu J, Helgeson BE, Cao Q, Prensner JR, Rubin MA, Shah RB, Mehra R, Chinnaiyan AM, Michigan Center for Translational Pathology, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI

TMPRSS2-ERG gene fusions are the predominant molecular subtype of prostate cancer. Here, we explored the role of TMPRSS2-ERG gene fusion product using in vitro and in vivo model systems. Transgenic mice expressing the ERG gene fusion product under androgen-regulation develop mouse prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. Introduction of the ERG gene fusion product into primary or immortalized benign prostate epithelial cells induced an invasion-associated transcriptional program but did not increase cellular proliferation or anchorage-independent growth. These results suggest that TMPRSS2-ERG may not be sufficient for transformation in the absence of secondary molecular lesions. Transcriptional profiling of ERG knockdown in the TMPPRSS2-ERG-positive prostate cancer cell line VCaP revealed decreased expression of genes over-expressed in prostate cancer versus PIN and genes overexpressed in ETS-positive versus -negative prostate cancers in addition to inhibiting invasion. ERG knockdown in VCaP cells also induced a transcriptional program consistent with prostate differentiation. Importantly, VCaP cells and benign prostate cells overexpressing ERG directly engage components of the plasminogen activation pathway to mediate cellular invasion, potentially representing a downstream ETS target susceptible to therapeutic intervention. Our results support previous work suggesting that TMPRSS2-ERG fusions mediate invasion, consistent with the defining histologic distinction between PIN and prostate cancer.

Role of the TMPRSS2-ERG Gene Fusion in Prostate Cancer

TMPRSS2-ERG gene fusions are the predominant molecular subtype of prostate cancer. Here, we explored the role of TMPRSS2-ERG gene fusion product using in vitro and in vivo model systems. Transgenic mice expressing the ERG gene fusion product under androgen-regulation develop mouse prostatic intraepithelial neoplasia (PIN), a precursor lesion of prostate cancer. Introduction of the ERG gene fusion product into primary or immortalized benign prostate epithelial cells induced an invasion-associated transcriptional program but did not increase cellular proliferation or anchorage-independent growth. These results suggest that TMPRSS2-ERG may not be sufficient for transformation in the absence of secondary molecular lesions. Transcriptional profiling of ERG knockdown in the TMPPRSS2-ERG-positive prostate cancer cell line VCaP revealed decreased expression of genes over-expressed in prostate cancer versus PIN and genes overexpressed in ETS-positive versus -negative prostate cancers in addition to inhibiting invasion. ERG knockdown in VCaP cells also induced a transcriptional program consistent with prostate differentiation. Importantly, VCaP cells and benign prostate cells overexpressing ERG directly engage components of the plasminogen activation pathway to mediate cellular invasion, potentially representing a downstream ETS target susceptible to therapeutic intervention. Our results support previous work suggesting that TMPRSS2-ERG fusions mediate invasion, consistent with the defining histologic distinction between PIN and prostate cancer.