Late-stage Prostate Cancer Research Articles

Abstract 1630: Dysregulation of Hippo pathway in metastatic prostate cancer

Abstract Background: Prostate cancer (PCa) is prevalently diagnosed and more than 80% of late stage PCa patients develop bone metastases. We hypothesized that the selective lodging of the PCa cells in the bone can be contributed by increased RANK-mediated cell signaling network. Cancer cell-derived RANKL orchestrates a complex bone-remodeling program facilitating PCa bone colonization through the activation of specific transcription factors (TFs) promoting PCa-bone microenvironment interaction through increased epithelial to mesenchymal transition (EMT), cell growth, and osteomimicry. Overexpression of RNAKL in indolent LNCaP cells drives cancer bone and soft tissue metastases. Methods and Results: Microarray gene expression was performed to compare RANKL- and Neo-vector- transfected LNCaP cells, LNCaPRANKL and LNCaPneo, respectively. qRT-PCR and western blot were performed to confirm the expression of RANKL-targeted genes in these cells. We found the new extracellular diffusible signal mediated by RANKL, regulating Hippo pathway in the aggressive PCa cells. Recombinant RANKL-treated indolent PCa cells, LNCaP and LAPC4, were shown to gain increased aggressiveness. The RANKL-treated indolent PCa cells exhibited down-regulation of Hippo pathway core kinases (Mst1/2 and Lats1/2) but up-regulation of YAP/TAZ expressions. By deleting downstream effectors of Hippo pathway, YAP/TAZ, interrupted the aggressive PCa cells to migrate, proliferate and invade and reversed their EMT phenotype. Inhibitions of YAP/TAZ in aggressive PCa cells via genetic knockdown approach resulted in E-cadherin increase and Vimentin decrease. With the identification of crosstalk between Hippo and TGF-β, or Wnt pathways, this opened up a new network and targeting opportunity for PCa bone metastasis. The metastatic PCa cells with YAP/TAZ deletions were illustrated for sensitization to other therapeutic agents such as TGF-β inhibitors. These results prompted us to study the mechanism, validate the functional significance, and devise potential targeting strategies of Hippo pathway in PCa bone metastasis. Conclusions: Our results showed that dysregulated Hippo pathway in the aggressive PCa cells. With deletions of YAP/TAZ, PCa metastatic abilities were inhibited. Our findings might explain why PCa cells prefer bone as primary metastatic sites. In addition, targeting YAP/TAZ could be a potential therapy for PCa bone metastasis. Citation Format: Jen-Ming Huang, Gina C-Y Chu, Hyung L. Kim, Haiyen E. Zhau, Leland W.K. Chung. Dysregulation of Hippo pathway in metastatic prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1630.

261. Gain-of-Function Effect Augments Therapeutic Efficacy of CpG-STAT3 Anti-Sense Oligonucleotide Against Castration-Resistant Prostate Cancers

The treatment for castration-resistant prostate cancers (CRPC) remains a clinical challenge. STAT3 expression level correlates with poor prognosis in prostate cancer patients and resistance to hormone therapy. In addition to oncogenic role of STAT3 in cancer cells, it is also commonly activated in the prostate tumor microenvironment. Therefore, STAT3 is a promising target targeting STAT3 is expected to generate dual effects; anti-tumor activity and anti-immunosuppression in CRPC. Targeting transcriptions factors for cancer therapy has not been successful due to the lack of enzymatic activity. Recent advances in oligonucleotide-based therapy using anti-sense oligonucleotide (ASO) targeting STAT3 had shown promising results in phase I clinical trials in various human cancers, including liver metastasis. However, the limitation fo these reagents remained targeting We previously demonstrated that Toll-like Receptor 9 (TLR9) is up-regulated in prostate cancer stem-like cells in the late-stage prostate cancer. STAT3 signaling is activated in TLR9+ prostate cancer cells and tumor associated immune cells. Here, we describe a new molecule that enables specific delivery of STAT3 ASO to TLR9+ tumor and immune by linking STAT3 ASO to CpG ODN, TLR9 ligand. CpG-STAT3 ASO was quickly and efficiently internalized by human and mouse prostate cancer cell lines showing cytoplasmic localization after 15 minutes after incubation in vitro. Human and mouse TLR9+ immune cells; DCs, Macrophages and B cells were able to efficiently uptake CpG-STAT3 ASO in vitro. In a biodistribution experiment, CpG-STAT3 ASOCy3 was effectively internalized by TLR9+ myeloid cells in various organs including lymph node and bone marrow. Treatment with CpG-STAT3 ASO conjugates in vitro decreased the expression of STAT3 in TLR9+ human (DU145 and LNCaP-TLR9) and mouse (RM1 and RM9) prostate cancer cells more efficiently than unconjugated STAT3 ASO. Notably, CpG-STAT3 ASO showed significantly augmented cytotoxicity in prostate cancer cells compared to STAT3 ASO. In a preliminary anti-tumor efficacy study using syngeneic mouse model, repeated intra-tumoral injections of CpG-STAT3 ASO reduced the tumor growth of RM9. In contrast, the inhibitory effect of STAT3 ASO was transient leading to tumor relapse after the initial treatments. Noteworthy, CpG-STAT3 ASO reduced tumor growth and STAT3 expression in the untreated tumors in the contra-lateral site. Moreover, CpG-STAT3 ASO, but not the unconjugated STAT3 ASO, reduced activation of STAT3 in infiltrated myeloid-derived suppressor cells (MDSC) (CD11b+Gr1+) in both tumors and the percentage of T reg (CD4+FoxP3+) cells in the tumor draining lymph nodes. These results implicate that only when combined with concomitant TLR9 stimulation STAT3 inhibition can systemically overcome tumor immune tolerance. Overall, these results suggest that CpG-STAT3 ASO conjugate holds a great promise for an anti-cancer therapy of CRPC by simultaneously targeting STAT3 signaling in TLR9+ prostate cancer cells and tumor-associated immune cells.

MET expression during prostate cancer progression.

Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression.Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization.None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, χ2), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; χ2), with MET polysomy in 8/13 (61%) evaluable cases.In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc.

Interaction of EGFR to δ-catenin leads to δ-catenin phosphorylation and enhances EGFR signaling.

Expression of δ-catenin reportedly increases during late stage prostate cancer. Furthermore, it has been demonstrated that expression of EGFR is enhanced in hormone refractory prostate cancer. In this study, we investigated the possible correlation between EGFR and δ-catenin in prostate cancer cells. We found that EGFR interacted with δ-catenin and the interaction decreased in the presence of EGF. We also demonstrated that, on one hand, EGFR phosphorylated δ-catenin in a Src independent manner in the presence of EGF and on the other hand, δ-catenin enhanced protein stability of EGFR and strengthened the EGFR/Erk1/2 signaling pathway. Our findings added a new perspective to the interaction of EGFR to the E-cadherin complex. They also provided novel insights to the roles of δ-catenin in prostate cancer cells.

Texas Native Plants Yield Compounds with Cytotoxic Activities against Prostate Cancer Cells.

There remains a critical need for more effective therapies for the treatment of late-stage and metastatic prostate cancers. Three Texas native plants yielded three new and three known compounds with antiproliferative and cytotoxic activities against prostate cancer cells with IC50 values in the range of 1.7-35.0 μM. A new sesquiterpene named espadalide (1), isolated from Gochnatia hypoleuca, had low micromolar potency and was highly effective in clonogenic assays. Two known bioactive germacranolides (2 and 3) were additionally isolated from G. hypoleuca. Dalea frutescens yielded two new isoprenylated chalcones, named sanjuanolide (4) and sanjoseolide (5), and the known sesquiterpenediol verbesindiol (6) was isolated from Verbesina virginica. Mechanistic studies showed that 1-4 caused G2/M accumulation and the formation of abnormal mitotic spindles. Tubulin polymerization assays revealed that 4 increased the initial rate of tubulin polymerization, but did not change total tubulin polymer levels, and 1-3 had no effects on tubulin polymerization. Despite its cytotoxic activity, compound 6 did not initiate changes in cell cycle distribution and has a mechanism of action different from the other compounds. This study demonstrates that new compounds with significant biological activities germane to unmet oncological needs can be isolated from Texas native plants.

Ex vivo propagation and characterization of circulating tumor cell clusters from late stage prostate cancer patients.

314 Background: Cells shed by a tumor into the bloodstream upon metastasis present an opportunity to study the properties of these cells for biomarker discovery, drug development, and personalized therapy. Enrichment and expansion of circulating tumor cells (CTCs) in culture enables molecular characterization not readily available for rare cells. Here, we demonstrate the successful identification and culture of rare circulating tumor cell clusters from peripheral blood of metastatic castration resistant prostate cancer patients (mCRPC). Methods: The approach to isolate CTC clusters relies on nucleated cell enrichment via Ficoll-based centrifugation from 15 mL of peripheral blood. A collagen-based cell-binding substrate was used to capture cell clusters with preferential affinity for collagen (i.e. epithelial-like cells). Hypoxic and pressurized culturing conditions were used to maintain and propagate adhered CTC clusters. After 7 days in culture, immunofluorescence imaging, qPCR and/or mRNAseq were performed on CTC clusters that had formed colonies. Results: Blood samples processed within 3 hours from time of draw yielded a 25% success rate (4/16) for identifying PSMA+/EpCAM+/CD45- CTC colonies. Morphologically distinct CTC colonies cultivated at 1% oxygen and increased hydrostatic pressure (2 PSI) were positive for PSMA and EpCAM staining as observed via confocal microscopy, with individual colonies ranging from approximately 50 to 1000 in cell number. Colonies consistently revealed white blood cell contaminants (T-cells and dendritic cells, 10 to 30% of colony) that did not appear to perturb colony growth. CTC colonies were collected for qPCR analysis and mRNAseq via laser-capture microdissection and/or micropipetting. Genomic analysis revealed differential gene expression patterns between CTC colonies cultured from the same patient sample, with a subset expressing neuroendocrine like signatures (AURKA), stem cell markers (SOX2, OCT4), and immuno-oncology relevant markers (PD-L1, CTLA4). Conclusions: In conclusion, we show that viable CTCs are amenable to ex vivo culture, providing both functional and molecular insights into a sub-population of CTCs with propagating potential. The presence of T-cells and dendritic cells on CTC colonies warrants further investigation, and may provide unique insights into immune-tumor interactions. The culturing platform is currently being evaluated as a research tool for biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making.

Genomic predictors for treatment of late stage prostate cancer.

In spite of the development of new treatments for late stage prostate cancer, significant challenges persist to match individuals with effective targeted therapies. Genomic classification using high-throughput sequencing technologies has the potential to achieve this goal and make precision medicine a reality in the management of men with castrate-resistant prostate cancer. This chapter reviews some of the most recent studies that have resulted in significant progress in determining the landscape of somatic genomic alterations in this cohort and, more importantly, have provided clinically actionable information that could guide treatment decisions. This chapter reviews the current understanding of common alterations such as alterations of the androgen receptor and PTEN pathway, as well as ETS gene fusions and the growing importance of PARP inhibition. It also reviews recent studies that characterize the evolution to neuroendocrine tumors, which is becoming an increasingly important clinical problem. Finally, this chapter reviews recent innovative studies that characterize the compelling evolutionary history of lethal prostate cancer evidenced by polyclonal seeding and interclonal cooperation between metastasis and the importance of tumor clone dynamics measured serially in response to treatment. The genomic landscape of late stage prostate cancer is becoming better defined, and the prospect for assigning clinically actionable data to inform rationale treatment for individuals with this disease is becoming a reality.

Abstract A193: Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice

Abstract Despite multiple new treatment options for patients with castrate-resistant prostate cancer (CRPC), this is still an area of un-met need. Prostate specific membrane antigen (PSMA) is highly expressed in prostate cancer and expression is maintained in CRPC, making it an attractive target for late stage prostate cancer. We have developed a novel full-length human IgG bispecific antibody that binds to human and monkey PSMA and CD3 and results in T cell activation via the CD3 complex in the presence of PSMA-expressing tumors. In cell-based assays, our PSMAxCD3 bispecific resulted in target cell-dependent activation and cytokine release by human T cells, and efficient redirected T cell lysis of prostate tumor cells. Of note, in the absence of PSMA-expressing tumor cells, no T cell activation was seen. Our bispecific antibody also resulted in redirected killing by cynomolgus monkey T cells, enabling pre-clinical toxicity studies. To examine bispecific anti-tumor efficacy in vivo, we developed xenogenic and syngeneic tumor models. In studies with human tumor cells, NOD SCID IL2R gamma deficient (NSG) mice were co-implanted with human peripheral blood mononuclear (PBMC) cells plus human prostate tumor cells (22Rv1 or C4-2), and the mice treated immediately with PSMAxCD3 i.p. Significant inhibition of tumor growth was observed against both human prostate tumors with dose levels as low as 0.004mg/kg. Importantly, PSMAxCD3 bispecific antibodies could also suppress growth of established C4-2 tumors when PBMCs were transferred i.p.. Our PSMAxCD3 bispecific was also tested in mice engrafted with human hematopoietic CD34+ stem cells. Newborn SIRPα BALB/c-Rag2null IL2rγnull (BRG) pups were engrafted with hCD34+ fetal liver cells as hematopoietic progenitor cells, which gave rise to human T, B, and NK cells, as well as granulocytes, monocytes, and DCs. Once engraftment was achieved (3 months later), mice were given C4-2 tumor cells subcutaneously. Eight days after tumor implantation, mice were treated with PSMAxCD3 (0.4mg/kg) followed by twice weekly doses. The PSMAxCD3 bispecific reduced tumor growth in this model. To examine the effects of our lead-candidate bispecifics in an immune-competent model, we engineered mice to express both human CD3 and PSMA in the corresponding murine loci. T cells from these mice were efficiently activated by antibodies to human CD3, and the expression of human PSMA allowed assessment of effects on normal tissues that may express PSMA. T cell activation, proliferation and cytokine production in response to our bispecific antibodies were examined in the absence of tumor as well as the absence of target. To assess anti-tumor efficacy in this model, a murine prostate cancer line expressing human PSMA was generated and tumors cells were implanted s.c. into these mice. Our bispecific antibodies significantly suppressed tumor growth when mice were treated at the time of implantation and also when treatment was delayed until tumors were established. Moreover, T cells were still present at normal levels in the spleen of mice treated with our bispecific at the end of the experiment and the treatment was well-tolerated. Taken together, these studies show potent anti-tumor activity of our PSMAxCD3 bispecific in several different tumor models and also demonstrate that treatment was well-tolerated in mice expressing human PSMA and human CD3. Citation Format: Alison Crawford, Kristin Vazzana, Jeffrey VanValkenburgh, Lauric Haber, Jennifer Principio, Cagan Gurer, Kara Olson, Eric Smith, Gavin Thurston, Jessica R. Kirshner. Fully human bispecific antibodies induce potent anti-tumor effects against prostate tumors in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A193.

Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer.

During the past 10 years, preclinical studies implicating sustained androgen receptor (AR) signalling as the primary driver of castration-resistant prostate cancer (CRPC) have led to the development of novel agents targeting the AR pathway that are now in widespread clinical use. These drugs prolong the survival of patients with late-stage prostate cancer but are not curative. In this Review, we highlight emerging mechanisms of acquired resistance to these contemporary therapies, which fall into the three broad categories of restored AR signalling, AR bypass signalling and complete AR independence. This diverse range of resistance mechanisms presents new challenges for long-term disease control, which may be addressable through early use of combination therapies guided by recent insights from genomic landscape studies of CRPC.

A basal stem cell signature identifies aggressive prostate cancer phenotypes

Evidence from numerous cancers suggests that increased aggressiveness is accompanied by up-regulation of signaling pathways and acquisition of properties common to stem cells. It is unclear if different subtypes of late-stage cancer vary in stemness properties and whether or not these subtypes are transcriptionally similar to normal tissue stem cells. We report a gene signature specific for human prostate basal cells that is differentially enriched in various phenotypes of late-stage metastatic prostate cancer. We FACS-purified and transcriptionally profiled basal and luminal epithelial populations from the benign and cancerous regions of primary human prostates. High-throughput RNA sequencing showed the basal population to be defined by genes associated with stem cell signaling programs and invasiveness. Application of a 91-gene basal signature to gene expression datasets from patients with organ-confined or hormone-refractory metastatic prostate cancer revealed that metastatic small cell neuroendocrine carcinoma was molecularly more stem-like than either metastatic adenocarcinoma or organ-confined adenocarcinoma. Bioinformatic analysis of the basal cell and two human small cell gene signatures identified a set of E2F target genes common between prostate small cell neuroendocrine carcinoma and primary prostate basal cells. Taken together, our data suggest that aggressive prostate cancer shares a conserved transcriptional program with normal adult prostate basal stem cells.

2525 ASPIRE-PCa: Initial findings from a prospective, global observational study of men with late-stage prostate cancer

2525 ASPIRE-PCa: Initial findings from a prospective, global observational study of men with late-stage prostate cancer

Abstract LB-110: Expression of multiple androgen receptor splice variants in late stage prostate cancer

Abstract The androgen receptor (AR) remains a key driver of disease progression even with the advent of new therapeutic agents such as abiraterone (A) and enzalutamide (E). Relapse after treatment with A or E has been associated with the emergence of tumour cells expressing AR splice variants, and at least one mutation in AR has been associated with resistance to E. We have investigated the expression of AR splice variants in late stage prostate cancers using Nanostring (nCounter®) to quantify the levels of AR transcripts and by IHC using antibodies targeting the N- and C-terminal AR domains. We also examined the expression of putative AR regulated genes found to be expressed in prostate cancer reported in publically available expression data. Formalin fixed paraffin embedded tissue from 38 commercially sourced (TriStar Technology Group; Asterand; Avaden Biosciences) late stage prostate cancers including 10 castrate resistant prostate cancers (CRPC), were confirmed by pathology review. Total mRNA was isolated from a single 5μm section and gene expression from nCounter® analysis was expressed as normalised Log2 values. Immunohistochemical analysis of AR expression was determined by a pathologist using a H score. Our analysis of AR splice variant expression, relative to a Nanostring detection threshold based on housekeeping genes, has shown the presence of more than one constitutively active AR splice variant in individual tumours. ARV7 was detected in 4/10 CRPC and always found together with ARV1, AR45, and the constitutively active ARV12. All of the CRPC that were negative for ARV7 expressed AR45 and ARV12. Unsupervised clustering of 507 putative AR regulated genes revealed three molecular subgroups among the 38 prostate tumours. One of these subgroups was characterised by the presence of 12/14 AR splice variants including ARV7. Of the other two subgroups, one contained AR45, the other ARV12. Pathway analysis of genes that were expressed to a higher level (>1.5 Log2 fold) in the ARV7 positive compared to ARV7 negative tumours were predominantly associated with cell cycle and proliferation functions. Our data suggests complexity in the molecular landscape of prostate cancer associated with AR splice variants, and that multiple AR splice variants may be of importance to identify patients likely to relapse on treatment with emerging therapy. Citation Format: Zara Ghazoui, Emma Jones, Margaret Veldman-Jones, Vivien N. Jacobs, Neil R. Smith, Michele Johnstone, J. Carl Barrett, Elizabeth A. Harrington, Paul Elvin. Expression of multiple androgen receptor splice variants in late stage prostate cancer. [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 LB-110. doi:10.1158/1538-7445.AM2015-LB-110

Abstract 1727: DNA-binding domain as an alternative target site on androgen receptor for prostate cancer therapy: Pre-clinical in vivo study of a novel small molecule for drug development

Abstract Androgen receptor (AR) is a key regulatory transcription factor in the development of prostate cancer (PCa). Common treatments for PCa include hormone therapy, radiotherapy and surgery. However, many PCa patients develop resistance towards hormone therapies in later stages of PCa. There is growing evidence suggesting that AR splice variants render PCa treatments that target the LBD, such as enzalutimide, ineffective. Our objective is to develop a drug to treat recurrent and metastatic PCa by targeting the DNA Binding Domain (DBD). Through in silico docking prediction and in vitro bioassays, we have identified a novel small molecule - VPC-14449 (4-(4-(4,5-bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine) - that may inhibit AR activation through targeting DBD. In order to bring VPC -14449 into the clinic, we conducted preclinical in vivo studies to determine both toxicity and efficacy. CD1 mice were treated with 25, 50, and 100 mg/kg of VPC-14449 I.P. twice daily for four weeks to determine the maximum tolerable dose. During this time, we assessed the signs of physical toxicity, behavioral changes, and gross manifestations of stress. At the end of the experiment, the mice were euthanized and organs were collected for further immunohistochemical analysis. Furthermore, we assessed the in vivo therapeutic effects of VPC-14449 in different nude mouse xenograft models including LNCaP, MR49-F, and C4-2. Body weight, tumor volume (π/6 x length x width x height) and PSA were measured weekly. LNCaP xenograft mice were castrated when PSA level reached 25 ng/ml. Mice were randomly assigned to one of the three treatment groups (VPC-14449 (100mg/kg), enzalutimide (10 mg/kg), or vehicle) when PSA level returned to 25 ng/ml (for LNCaP xenografts) or when the tumor sizes reached 100mm3(for MR49-F and C4-2 xenografts). The mice were treated I.P. twice daily for four weeks before euthanization. Toxicity studies revealed no signs of physical toxicity, behavioral changes, and gross manifestations of stress in animals treated with up to 100 mg/kg dose of VPC-14449. Efficacy studies revealed that VPC-14449 effectively suppresses tumor growth in LNCaP, MR49 and C4-2 xenografts with P value <0.01, <0.05 and < 0.001 respectively, when compared to control. VPC-14449 also significantly reduced PSA levels in LNCaP xenografts (p value <0.01). The results suggest that VPC-14449 is effectively suppressing PCa tumor growth even in androgen insensitive and MDV resistant PCa cell lines in vivo. Citation Format: Mohamed DH Hassona, Leslie G. Chan, Gray R. Meckling, Huifang Li, Eric LeBlanc, Fuqiang Ban, Artem Cherkasov, Paul S. Rennie, Emma Guns. DNA-binding domain as an alternative target site on androgen receptor for prostate cancer therapy: Pre-clinical in vivo study of a novel small molecule for drug development. [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 1727. doi:10.1158/1538-7445.AM2015-1727

Abstract 931: Enrichment and characterization of propagating circulating tumor cells from late stage prostate and pancreatic cancer patients

Abstract Capturing cells shed by a tumor into the bloodstream represents an opportunity to study the properties of these cells for biomarker discovery in support of drug development and personalized therapy. Enrichment and expansion of circulating tumor cells (CTCs) in culture enables molecular characterization not readily available for rare cells. We report on a primary cell culture platform to enrich and expand rare circulating cells from the blood of patients with metastatic castration resistant prostate cancer and pancreatic ductal adenocarcinoma. The approach relies on tumor cell enrichment via cell-binding substrates comprised of a collagen-based hydrogel for CTC capture, and hypoxic culturing conditions to maintain and propagate subset of these cells with tumor-initiating potential. Here, we demonstrate the successful identification and expansion of rare circulating tumor cells from peripheral blood and leukapheresed blood samples from prostate and pancreatic cancer patients, respectively. Upon successful culture of samples for a period of 5 to10 days after plating, immunofluorescence imaging was performed. Morphologically distinct CTC colonies positive for PSMA, EpCAM and cytokeratin staining were observed, with individual colonies ranging from approximately 50 to 1000 in cell number. Interestingly, colonies consistently revealed white blood cell contaminants (T-cells and dendritic cells) that did not appear to perturb colony growth. Targeted DNA sequencing was performed on pancreatic CTC colonies from six patients using a genetic panel covering 237 genes. We achieved 100-200X coverage for each patient sample and present a preliminary genetic characterization of their CTCs in culture. Our results show unique genetic signatures indicative of aggressive forms of pancreatic ductal adenocarcinoma based on SNP/INDEL detection, CNV analysis and gene fusion predictions. In summary, cultured CTCs had 20-30X more variants than corresponding white blood cell controls, with mutations observed in MLL2, ARID1A, BRAF, KRAS and BRCA2. CNV analysis and gene fusion predictions revealed previously unreported biomarker candidates. In conclusion, we show that viable CTCs are amenable to ex vivo culture, providing both functional and molecular insights into a sub-population of CTCs with propagating potential. The presence of T-cells and dendritic cells on CTC colonies warrants further investigation, and may provide unique insights into immune-tumor interactions. The culturing platform is currently being evaluated as a research tool for biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making. Citation Format: James I. Lim, Charles J. Ryan, Thomas Krahn, Nikolas H. Stoecklein, Johannes Fischer, Bruce Adams. Enrichment and characterization of propagating circulating tumor cells from late stage prostate and pancreatic cancer patients. [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 931. doi:10.1158/1538-7445.AM2015-931

Impact of age, race and socio-economic status on temporal trends in late-stage prostate cancer diagnosis in Florida

Individual-level data from the Florida Cancer Data System (1981–2007) were analyzed to explore temporal trends of prostate cancer late-stage diagnosis, and how they vary based on race, income and age. Annual census-tract rates were computed for two races (white and black) and two age categories (40–65, >65) before being aggregated according to census tract median household incomes. Joinpoint regression and a new disparity statistic were applied to model temporal trends and detect potential racial and socio-economic differences. Multi-dimensional scaling was used as an innovative way to visualize similarities among temporal trends in a 2-D space. Analysis of time-series indicated that late-stage diagnosis was generally more prevalent among blacks, for age category 40–64 compared to older patients covered by Medicare, and among classes of lower socio-economic status. Joinpoint regression also showed that the rate of decline in late-stage diagnosis was similar among older patients. For younger patients, the decline occurred at a faster pace for blacks with rates becoming similar to whites in the late 1990s, in particular for higher incomes. Both races displayed distinct spatial patterns with higher rates of late-stage diagnosis in the Florida Panhandle for whites whereas high rates clustered in South-eastern Florida for blacks.

TLR9 signaling through NF-κB/RELA and STAT3 promotes tumor-propagating potential of prostate cancer cells.

Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew in vivo. Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients.

Geographically-weighted regression analysis of percentage of late-stage prostate cancer diagnosis in Florida

This study assessed spatial context and the local impacts of putative factors on the proportion of prostate cancer diagnosed at late-stages in Florida during the period 2001–2007. A logistic regression was performed aspatially and by geographically-weighted regression (GWR) at the nodes of a 5 km spacing grid overlaid over Florida and using all the cancer cases within a radius of 125 km of each node. Variables associated significantly with high percentages of late-stage prostate cancer included having comorbidities, smoking, being Black and living in census tracts with farmhouses. Having private or public insurance, being married or diagnosed in a for-profit facility, as well as living in census tracts with high household income reduced significantly this likelihood. Geographically-weighted regression allowed the identification of areas where the local odds ratio is significantly different from the ratio estimated using aspatial regression (State-level). For example, the local odds ratios for the comorbidity covariates were significantly smaller than the State-level odds ratio in Tallahassee and Pensacola, while they were significantly larger in Palm Beach. This emphasizes the need for local strategies and cancer control interventions to reduce the percentage of prostate cancer diagnosed at late-stages and ultimately eliminate health disparities.

698. Eliminating TLR9+ Prostate Cancer Stem Cells In Vivo Using NF-kB/RELA- or STAT3-Targeting CpG-siRNA Conjugates

Treatment of hormone-refractory and frequently reoccurring prostate cancers is a major clinical challenge. Here, we demonstrate that Toll-like Receptor 9 (TLR9) is commonly upregulated in late-stage prostate cancers and provides a potential therapeutic target. As a sensor of immunogenic cell death, TLR9 bridges intra-prostatic inflammation to cancer stem cell phenotype. Our limited dilution/serial transplantation experiments in vivo demonstrate that TLR9 is essential for prostate cancer cells’ potential to propagate and self-renew. In addition, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of prostate cancer cells. Genome-wide transcriptional analysis of cancer cells isolated from xenotransplanted prostate tumors revealed a unique TLR9-dependent gene expression signature. Further analysis of the TLR9 downstream signaling indicated that tumorigenic transcription factors NF-kB/RELA and STAT3 cooperate to orchestrate expression of stem cell-related genes. Both RELA and STAT3 bound and co-regulated promoters of NKX3.1 and KLF4 prostate cancer stem cell-related genes. We further demonstrated the feasibility of targeting prostate cancer-propagating potential in vivo by TLR9-targeted siRNA delivery using CpG-siRNA conjugates. Local administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates, inhibited tumor growth and cancer cell clonogenic potential in two xenotransplanted prostate cancer models. Selective elimination of tumor-propagating cells using TLR9-targeted blockade of NF-?B/RELA and STAT3 signaling has potential for clinical translation to benefit patients with late-stage prostate cancers.

Free Fatty Acid Receptor 4 (FFA4) Mediates the Inhibitory Effects of Omega‐3 Fatty Acids on Growth Factor‐mediated Proliferation and Migration in Human Prostate Cancer Cells

Both in vitro and in vivo experiments have shown that omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have promising inhibitory effects on prostate cancer cell growth and invasion. Omega-3 fatty acids act via several potential molecular mechanisms that include their action as agonists at free fatty acid receptors (FFARs), which are G protein-coupled receptors. Two FFARs that bind omega-3 fatty acids, FFA1 and FFA4, are expressed in DU145 and PC-3 cells, two late-stage human prostate cancer cell lines. Our group has shown that both EPA and TUG-891, a synthetic, highly selective FFA4 (formerly GPR120) agonist, completely inhibit lysophosphatidic acid (LPA)- and epidermal growth factor (EGF)-mediated proliferation and migration in DU145 and PC-3 cells. The FFA4 agonists also significantly inhibit proliferation induced by fetal bovine serum. The inhibitory effects are readily reversed upon removal of TUG-891 or EPA., demonstrating that the FFA4 agonists must be continually present in order to inhibit proliferation To further confirm the involvement of FFA4, we utilized siRNA to decrease FFA4 expression in DU145 and PC-3 cells. We found that FFA4 knockdown eliminates the inhibitory effects of EPA and TUG-891 on proliferation and migration in both cell lines, in response to both LPA and EGF. Our data suggest that FFA4 is a promising therapeutic target in prostate cancer.