Metastatic Prostate Cancer Cells Research Articles

GRP78 modulates cell adhesion markers in prostate Cancer and multiple myeloma cell lines

BackgroundGlucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment.MethodsN-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells.ResultsGRP78 KD led to concomitant downregulation of N-cad in both tumors types. In PC3 cells, GRP78 KD significantly decreased E-cadherin (E-cad) expression likely associated with the induction in TGF-β1 expression. Furthermore, GRP78 KD also triggered drastic changes in PC3 cells morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cad expression.ConclusionThis work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche.

Humanization of the Prostate Microenvironment Reduces Homing of PC3 Prostate Cancer Cells to Human Tissue-Engineered Bone

The primary tumor microenvironment is inherently important in prostate cancer (PCa) initiation, growth and metastasis. However, most current PCa animal models are based on the injection of cancer cells into the blood circulation and bypass the first steps of the metastatic cascade, hence failing to investigate the influence of the primary tumor microenvironment on PCa metastasis. Here, we investigated the spontaneous metastasis of PC3 human PCa cells from humanized prostate tissue, containing cancer-associated fibroblasts (CAFs) and prostate lymphatic and blood vessel endothelial cells (BVECs), to humanized tissue-engineered bone constructs (hTEBCs) in NOD-SCID IL2Rγnull (NSG) mice. The hTEBC formed a physiologically mature organ bone which allowed homing of metastatic PCa cells. Humanization of prostate tissue had no significant effect on the tumor burden at the primary site over the 4 weeks following intraprostatic injection, yet reduced the incidence and burden of metastases in the hTEBC. Spontaneous PCa metastases were detected in the lungs and spleen with no significant differences between the humanized and non-humanized prostate groups. A significantly greater metastatic tumor burden was observed in the liver when metastasis occurred from the humanized prostate. Together, our data suggests that the presence of human-derived CAFs and BVECs in the primary PCa microenvironment influences selectively the metastatic and homing behavior of PC3 cells in this model. Our orthotopic and humanized PCa model developed via convergence of cancer research and tissue engineering concepts provides a platform to dissect mechanisms of species-specific PCa bone metastasis and to develop precision medicine strategies.

Suppression of Taxanes Cytotoxicity by Citrus Flavonoid Hesperetin in PPC-1 Human Prostate Cancer Cells.

More than half of prostate cancer patients use, in addition to conventional therapies, some kind of complementary medicine, including flavonoid-rich products. However, knowledge about the co-effects of flavonoids with cytotoxic chemotherapies is still rather poor. Therefore, this study was undertaken to assess the cytotoxic activity of flavonoids and their interactions with taxanes in human advanced prostate cancer cells. Cytotoxicity of different flavonoids and their effects on the efficacy of docetaxel and cabazitaxel were studied in the human metastatic prostate cancer cell line PPC-1, using MTT colorimetric assay. Both taxanes suppressed the viability of PPC-1 cells with IC50 values in the nanomolar range. Tested flavonoids exerted cytotoxic activity only at high micromolar concentrations or revealed no remarkable effect on cell survival. Simultaneous treatment of cells with taxanes and flavonoids baicalein, chrysin, luteolin, fisetin, quercetin, genistein or daidzein did not lead to any change in chemotherapy-induced cytotoxicity. However, simultaneous exposure of cells to hesperetin and taxanes resulted in 9.8- and 13.1-fold reduction in cytotoxicity of docetaxel and cabazitaxel, respectively. Flavonoid hesperetin remarkably suppressed the cytotoxic efficacy of taxanes in prostate cancer cells. Therefore, caution is required from prostate cancer patients who take hesperetin-containing oral supplements.

Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells.

Purvalanol and roscovitine are specific cyclin-dependent kinase (CDK) inhibitors, which have antiproliferative and apoptotic effects on various types of cancer. Although, the apoptotic accomplishment of purvalanol and roscovitine was elucidated at the molecular level, the underlying exact of drug-induced apoptosis through mitogen-activated protein kinase (MAPK) signaling still speculative. In addition, the role of CDK inhibitors in the downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated epithelial-mesenchymal transition (EMT) remains unclear. Here, we investigated the potential effect of each CDK inhibitors on cell proliferation, migration, and generation of reactive oxygen species due to the inhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. We reported that purvalanol and roscovitine induced mitochondria membrane potential loss-dependent apoptotic cell death, which was also characterized by activation of several caspases, cleavage of poly (ADP-ribose) polymerase-1 in DU145 and PC3 cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126, synergistically suppressed cell proliferation, and induced apoptotic action. Also, ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the downregulation of EMT processes via increasing the epithelial marker and decreasing mesenchymal markers through reduction of Wnt signaling regulators in DU145 cells. This study provides biological evidence about purvalanol and roscovitine have apoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell by activation of GSK3β signaling and inhibition of phosphoinositide-3-kinase/AKT (PI3K/AKT) pathways involved in the EMT process.

Enhanced Cancer Theranostics with Self-Assembled, Multilabeled siRNAs.

The integration of therapy and diagnostics, termed “theranostics”, has recently gained widespread utility in the development of new and improved therapeutics that effectively diagnose and treat diseases, such as cancer. In this study, the covalent attachment of multiple fluorescent labels (i.e., fluorescein isothiocyanate (FITC)) to a wide range of siRNAs, including those adopting linear, V- and Y-shape nanostructures, was successfully accomplished by solid-phase bioconjugation for monitoring cell uptake, co-localization, and biological activity in cell culture. The FITC-labeled higher-order V- and Y-shape siRNAs maintained the requisite hybrid stabilities and A-type helical structures for invoking RNAi activity. The FITC–siRNA hybrids with sense-strand modifiers enabled efficient mRNA knockdown (∼50–90%), which also translated to increased cell death (∼20–95%) in a bone metastatic prostate cancer cell line, over a 72 h incubation period. Significantly, the Y-shaped siRNA containing three FITC probes enhanced fluorescent signaling relative to the siRNA constructs containing single and double fluorophores while retaining potent knockdown and cell death effects post-transfection. Taken together, this data highlights the theranostic utility of the multilabeled FITC–siRNA constructs for potential cancer gene therapy applications.

SMC1A is associated with radioresistance in prostate cancer and acts by regulating epithelial-mesenchymal transition and cancer stem-like properties.

Prostate cancer is one of the most commonly diagnosed cancers and a pressing health challenge in men worldwide. Radiation therapy (RT) is widely considered a standard therapy for advanced as well as localized prostate cancer. Although this primary therapy is associated with high cancer control rates, up to one-third of patients undergoing radiation therapy becomes radio-resistant and/or has tumor-relapse/recurrence. Therefore, focus on new molecular targets and pathways is essential to develop novel radio-sensitizing agents for the effective and safe treatment of prostate cancer. Here, we describe functional studies that were performed to investigate the role of structural maintenance of chromosome-1 (SMC1A) in radioresistance of metastatic prostate cancer cells. Short hairpin RNA (shRNA) was used to suppress SMC1A in metastatic castration-resistant prostate cancer cells, DU145 and PC3. Clonogenic survival assays, Western blot, RT-PCR, and γ-H2AX staining were used to assess the effect of SMC1A knockdown on radiation sensitivity of these prostate cancer cells. We demonstrate that SMC1A is overexpressed in human prostate tumors compared to the normal adjacent tissue. SMC1A knockdown limits the clonogenic potential, epithelial-mesenchymal transition (EMT), and cancer stem-like cell (CSC) properties of DU145 and PC3 cells and enhanced efficacy of RT in these cells. Targeted inhibition of SMC1A not only plays a critical role in overcoming radio-resistance in prostate cancer cells, but also suppresses self-renewal and the tumor-propagating potential of x-irradiated cancer cells. We propose that SMC1A could be a potential molecular target for the development of novel radio-sensitizing therapeutic agents for management of radio-resistant metastatic prostate cancer.

ZIP9 but not the androgen receptor mediates testosterone-induced migratory activity of metastatic prostate cancer cells

LNCaP cells are derived from a metastatic lesion of human prostate adenocarcinoma. They express the classical androgen receptor (AR) and ZIP9, a Zn2+ transporter that also binds testosterone and mediates signaling by interacting with G-proteins.Our results show that LNCaP cells respond to testosterone by mobilizing their migratory machinery. Their exposure to testosterone triggers the formation of lamellipodia, reorganization of the actin cytoskeleton, phosphorylation of focal adhesion kinase (FAK) at Tyr925 and of paxillin at Tyr118, expression of matrix metalloproteinase 2 (MMP-2), and cell migration.Silencing ZIP9 expression by means of siRNA does not affect the responsiveness of the classical AR to testosterone; however, it prevents all of the testosterone effects described above: formation of lamellipodia cannot be induced, stimulation of FAK or paxillin phosphorylation or MMP-2 expression is prevented, and cell migration does not take place in the absence of ZIP9.The data presented show that testosterone/ZIP9 interactions might have not only physiological but also pathophysiological relevance. The fact that the migratory machinery of a metastatic prostate cancer cell line is activated exclusively through testosterone/ZIP9 and not through testosterone/AR interactions suggests that targeting specific inhibition of testosterone/ZIP9-mediated events might help in developing new therapeutic strategies against androgen-induced progression of prostate cancer.

Reduced Arginyltransferase 1 is a driver and a potential prognostic indicator of prostate cancer metastasis.

Most prostate cancer cases remain indolent for long periods of time, but metastatic progression quickly worsens the prognosis and leads to mortality. However, little is known about what promotes the metastasis of prostate cancer and there is a lack of effective prognostic indicators, making it immensely difficult to manage options for treatment or surveillance. Arginyltransferase 1 (Ate1) is the enzyme mediating post-translational protein arginylation, which has recently been identified as a master regulator affecting many cancer-relevant pathways including stress response, cell cycle checkpoints, and cell migration/adhesion. However, the precise role of Ate1 in cancer remains unknown. In this study, we found the occurrence of metastasis of prostate cancer is inversely correlated with the levels of Ate1 protein and mRNA in the primary tumor. We also found that metastatic prostate cancer cell lines have a reduced level of Ate1 protein compared to non-metastatic cell lines, and that a depletion of Ate1 drives prostate cancer cells towards more aggressive pro-metastatic phenotypes without affecting proliferation rates. Furthermore, we demonstrated that a reduction of Ate1 can result from chronic stress, and that shRNA-reduced Ate1 increases cellular resistance to stress, and drives spontaneous and stress-induced genomic mutations. Finally, by using a prostate orthotropic xenograft mouse model, we found that a reduction of Ate1 was sufficient to enhance the metastatic phenotypes of prostate cancer cell line PC-3 in vivo. Our study revealed a novel role of Ate1 in suppressing prostate cancer metastasis, which has a profound significance for establishing metastatic indicators for prostate cancer, and for finding potential treatments to prevent its metastasis.

Abstract A075: Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis?

Abstract Cancer is a disease of accumulated genetic mutations that alter a cell’s function. One of the common cellular functions affected in cancer cells is the apoptotic pathway. Apoptosis, also known as programmed cell death, is a regulatory biologic function controlled by protein interactions within the BCL-2 protein family. Normally, cells use this pathway to induce cell death when they detect an error in the cell’s functioning that cannot be fixed. In multiple types of cancers, including prostate cancer, there is often an excess of pro-survival BCL-2 proteins. Consequently, these cells have an inability to induce apoptosis, leading to an increase in cancer cell survival, sometimes even in the presence of chemotherapeutic chemicals. This research investigates the potential difference in BCL-2 proteins between metastatic prostate cancer cells and premetastatic prostate cancer cells. Specifically, changes in cancer cell response to chemotherapy after inhibiting antiapoptotic BCL-2 proteins via small-molecule inhibitors are being evaluated. We hypothesize that chemoresistant cancer cells are sensitized to BCL-2 inhibitors by first priming their mitochondria with a traditional chemotherapeutic agent. “Mitochondrial priming” is bringing the antiapoptotic BCL-2 proteins to their apoptotic threshold (1). To accomplish these aims, metastatic prostate cancer cell lines PC3, LnCap C4/2, and DU145 as well as a normal prostate cancer cell line, PNT2, were used. We first sought to compare the level of BCL-2 family proteins among the different cell types. PC3 cells have significantly higher levels of BCL-2, BCL-xL, and BAD as detected by Western blot and quantified by densitometry, corroborating findings from other labs. Next, cells were treated with 5uM etoposide or 100nM docetaxel for 24 hours or 48 hours, and viability was assessed by MTT and apoptosis via Annexin V-FITC. We found that cell viability was significantly decreased in LnCap and DU145 cells at 24 hours, which can be attributed specifically to apoptosis. Moreover, increasing amounts of apoptosis occurred after 48 hours of treatment. Comparatively, PNT2 and PC3 cells have significantly less apoptosis induced by either etoposide or docetaxel treatment. Apoptosis was further confirmed by caspase-3 activation as well as the ability to block apoptosis by pretreatment with the caspase inhibitor Boc-D-fmk in LnCap cells. Additionally, overexpression of BCL-xL in LnCap cells abrogated etoposide-induced apoptosis. Therefore BCL-xL is a potential target for priming. Next, we focused on enhancing the apoptotic effects of etoposide and docetaxel by combing their use with ABT-737, which is a pan BCL-2 antiapoptotic inhibitor. Evaluating the effects of these drugs on various prostate cancerous cell lines in comparison to the effects on a normal prostate cell line may provide insight into their clinical usefulness and relevance in combination treatments. Cells were treated with 0, 100nM, 500nM, 1uM, 2.5uM, or 5uM ABT-737 for 24 hours and apoptosis was assessed via Annexin V-FITC. Preliminary results suggest that on its own, ABT-737 has little effect on LnCap cells (~10%) and no effect on PC3, DU-145, or PNT2 cells. However, pretreating LnCap cells for 24 hours with etoposide as low as 100nM followed by 5 hours exposure to 500nM ABT-737 can induce twice as much apoptosis. These results suggest that etoposide “primes” cells and then the BCL-2 inhibitor pushes them over the edge towards apoptosis. Since less etoposide (100nM) in the combined treatment was effective, as opposed to 500nM when given alone, it may be possible to administer lower amounts of clinically established chemotherapeutics. However, preliminary results also show a ~30% increase in apoptosis in DU145 cells and 50% increase in the normal prostate cell line (PNT2) after combination treatment of 50nM docetaxel for 48 hours followed by 5 hours exposure to 1uM ABT-737. These results suggest that even though lower levels of chemotherapeutics may be effective at killing prostate cancer cells when paired with ABT-737, this combination treatment may be just as harmful to healthy prostate cells. Ongoing studies are also focused on combination treatments with ABT-199, which is selective for antiapoptotic BCL-xL. Reference: 1. Chonghalie TN, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011:334(6059):1129-33. Citation Format: Kaitlyn Walsh, Rachel Wilson, Mary E. McManamy, Victoria Del Gaizo Moore. Bcl-2 family proteins as drug targets; Can prostate cancer be primed for apoptosis? [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 A075.

Abstract B051: PDX-derived and patient-derived prostate cancer organoids as a clinically relevant platform for translational research

Abstract Metastatic castration-resistant prostate cancer (CRPC) is a genetically heterogeneous disease. Effective translational research demands a diverse and representative set of preclinical models. A major obstacle in the field is the limited capacity to culture prostate cancer-derived cells in vitro. Few cell lines exist. Those that are available do not well represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques developed in the Sawyers and Clevers laboratories have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but is restricted by limited availability of tissue and a success rate of <20% when establishing new lines. In the field of prostate cancer, patient-derived xenograft (PDX) lines are a model that broadly represents the clinical and genetic diversity of the disease. Like primary prostate tumors, PDXs have not been readily adaptable to in vitro culture (including the standard organoid method), making them poorly suited for mechanistic studies or high-throughput drug screens. Therefore, there is great interest in adapting the prostate cancer PDX model to an in vitro culture system. The LuCaP series of PDXs is a well-characterized set of ~40 diverse prostate cancer specimens. Using LuCaP PDXs, we systematically tested several modifications to the organoid culture method. This includes the addition of factors to modify metabolism, growth, and differentiation; removal of standard media components; and combinations thereof. We show that p38 is required for growth and survival of almost all the LuCaP organoids. Removal of the p38 inhibitor component from standard prostate organoid media improves initial growth and makes it possible to maintain many PDX-derived organoids over several generations. Additional modifications to individual LuCaP culture conditions improve growth. We successfully cultured 21 of 24 different LuCaPs attempted, for at least one generation, and 10 have been grown long-term. We show that PDX-derived organoids recapitulate the phenotype of the original PDX tumor, including AR-dependent growth. For example, we have shown that homologous recombination deficient LuCaP organoid responses to olaparib reflect the enhanced sensitivity seen for patients in clinical trials. Additionally, our method improves the success rate for culturing samples directly from patient biopsies. We show that 2 of 3 biopsy-derived organoid lines that we have established grow well in our conditions but not in standard published conditions. The third grows equally well in both. Further, our data suggest that p38 is a previously unappreciated target for CRPC treatment. Importantly, our work immediately and significantly increases the number of prostate cancer lines available to the community by making the LuCaP PDXs accessible to in vitro culture, and by improving the success rate for establishing organoids directly from patient samples. Citation Format: Michael L. Beshiri, Caitlin M. Tice, Crystal Tran, Holly M. Nguyen, Adam G. Sowalsky, Supreet Agarwal, Keith H. Jansson, Kerry McGowen, Aian Neil Alilin, JuanJuan Yin, Fatima H. Karzai, William L. Dahut, Eva Corey, Kathleen Kelly. PDX-derived and patient-derived prostate cancer organoids as a clinically relevant platform for translational research [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 B051.

Abstract B026: Axl may support dormancy of disseminated tumor cells by inducing cell clustering and decreasing proliferation of metastatic prostate cancer cells

Abstract While the majority of prostate cancer (PCa) patients are cured by radical prostatectomy and/or radiation, 10-15% of these men eventually develop recurrent disease in the form of lethal metastases. The most common site for PCa metastasis is the bone marrow. The time from primary tumor removal to recurrence can range from 1-2 years to decades, and constitutes a period of minimal residual disease in which cancer cells remain in the body but are clinically undetectable. It is thought that these disseminated tumor cells (DTCs) are able to maintain a state of cellular dormancy in the bone marrow in which they are not proliferating or dying, and may be the seeds for lethal metastases. By understanding the mechanism(s) that regulate dormancy, we may be able to prevent metastasis altogether. Our group has previously found that the secreted bone marrow factor Gas6 and the expression of its receptor Axl are associated with dormant DTCs in xenograft mouse models. Thus, we hypothesize that Gas6 signaling through Axl mediates dormancy. In this study, we sought to functionally support this hypothesis both in vitro and in human PCa tissue. Overexpression of Axl in the C42B PCa cell line decreased proliferation in vitro, and this was independent of Gas6 when Axl was highly overexpressed. Axl overexpression also led to aggregation of C42B cells, suggestive of a possible role in circulating tumor cell (CTC) clustering to survive the circulation and inhabit the bone marrow. Furthermore, immunohistochemical staining of a PCa tissue microarray showed almost no Axl positivity in cancer cells, regardless of Gleason score and other clinical outcomes. Negative staining was also observed in sections from liver, lymph node, and tibial metastases. Consistent with this, Axl expression correlates with decreased incidence of biochemical recurrence and increased overall survival in the GRID and TCGA databases, respectively. These results indicate an antiproliferative role for Axl in PCa, and suggest its temporal expression during the natural history of PCa. Ongoing work includes assessing the expression of Axl on patient CTCs and DTCs, and using xenograft mouse models of metastasis to analyze the effects of Axl overexpression and knockout in PCa cell lines. Citation Format: Haley D. Axelrod, Kenneth C. Valkenburg, Jessica L. Hicks, Angelo M. DeMarzo, Kenneth J. Pienta. Axl may support dormancy of disseminated tumor cells by inducing cell clustering and decreasing proliferation of metastatic prostate cancer cells [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 B026.

Circulating monocytes from prostate cancer patients promote invasion and motility of epithelial cells.

BackgroundRecruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor‐derived signals.MethodsBlood monocytes from advanced and stable PCa patients were cultured, and the conditioned media (CM) were collected and analyzed using standard invasion and wound closure assays to measure effects on invasion and motility of PCa tumor cells. We then identified the proteome profile of these monocytes using proteome array and ELISA.ResultsConditioned media from circulating monocytes in patients with metastatic prostate cancer (PCa‐M) increased invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte‐derived CM from metastatic castration‐resistant (mCRPC) patients presented high levels of chitinase‐3‐like 1 (CHI3L1, YKL‐40) when compared to patients with stable disease (PCa‐N) and healthy control individuals (HC). The only described receptor for CHI3L1, interleukin‐13 receptor α2 (IL‐13Rα2), was significantly up‐regulated in the human metastatic PCa cell line, ARCaPM. Accordingly, we observed that the activation of IL‐13Rα2 from PCa‐M CM increased the invasiveness of ARCaPM cells while siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa‐M patients.ConclusionsThus, we show that circulating monocytes from metastatic PCa patients exert a tumor‐promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.

RNA sequencing reveals upregulation of a transcriptomic program associated with stemness in metastatic prostate cancer cells selected for taxane resistance

Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.

Abstract 4260: Dynamic modeling of macrophage plasticity in bone metastatic prostate cancer

Abstract Tumor-associated macrophages, particularly those with an anti-inflammatory phenotype, have long been implicated in the progression of primary solid malignancies, including prostate cancer. Metastatic prostate cancer typically manifests in the bone where it induces painful osteogenic lesions that are incurable. Bone is naturally rich in myeloid-derived macrophages whose temporal polarization into pro- (M1) and anti-inflammatory (M2) phenotypes is critical for regulating the bone repair program mediated by bone-resorbing osteoclasts and bone-building osteoblasts. However, the dynamics of macrophage polarization in the context of bone metastatic prostate cancer are underexplored and difficult to address with traditional biologic approaches. To address the role of macrophage polarization in the context of bone metastatic prostate cancer, we first investigated the macrophage polarization temporal dynamics in normal bones and analyzed macrophage plasticity in vivo subsequent to intratibial injury. Bone marrows were isolated at several time points and profiled by flow cytometry for pro- and anti-inflammatory monocyte macrophage content. Contralateral tibias were analyzed for bone volume, osteoblast and osteoclast numbers. We then designed a mathematical model describing the different cell population dynamics in the bones. Generation of the model required testing a number of assumptions regarding macrophage polarization behavior. For example, it is unknown whether M1 resolves at the initial stages of bone injury repair through death or by repolarizing into M2. For each aspect, competing mechanistic assumptions were proposed and simulated by sets of ODEs. The best-fitting assumptions for each aspect were integrated into a single comprehensive ODE model to fully describe the dynamics of the bone resident cell populations during bone remodeling. This experimentally validated model is now being used to address how bone resident cells respond to metastatic prostate cancer cells. In conclusion, we have generated an ODE model that describes macrophage polarization over time during bone repair and how pro- and anti-inflammatory macrophages interact with bone stromal cells. The mathematical model predictions are in agreement with our biologic experiments in vivo and will allow us to interrogate how macrophage polarization impacts the behavior of metastatic prostate cancer cells in the bone microenvironment. Citation Format: Etienne A. Baratchart, Chen Hao Lo, Conor Lynch, David Basanta. Dynamic modeling of macrophage plasticity in bone metastatic 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 4260.

Abstract 2679: Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion

Abstract Trypsin-3 is a highly active protease that has recently been identified as a potential therapeutic target for the reduction of tumor growth and metastasis in prostate, breast and pancreatic cancers. Current trypsin inhibitors typically inhibit a broad range of different trypsin-like enzymes. Thus, novel approaches to identify trypsin-3 selective inhibitors are needed. Cyanobacteria produce a vast diversity of natural products with complex chemical structures many of which are potent protease inhibitors. We screened extracts of 140 cyanobacteria strains and discovered numerous strains showing selectivity towards trypsin-2 and -3 inhibition. We isolated a complex glycopeptide from one of these strains, which selectively inhibited human trypsin-2 and -3 with IC50 values of about 100 nM, while trypsin-1 was not inhibited. Importantly, we also found that this peptide inhibited invasion of aggressive and metastatic PC-3M prostate cancer cells though ECM preparation, while it did not affect the proliferation of the cells. Our results suggest that microbial natural products may offer a viable alternative source of potent and selective trypsin-3 inhibitors. Such inhibitors may be suitable, after further development, for targeting the mechanisms associated with the invasion and metastatic dissemination of cancer cells, i.e., for treatment of aggressive cancers. Citation Format: Hannu Koistinen, Matti Wahlsten, Muhammad N. Ahmed, Kaarina Sivonen, Matthias Nees, Ulf-Håkan Stenman, David P. Fewer. Cyanobacterial trypsin-3 inhibitor inhibits prostate cancer cell invasion [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 2679.

Abstract 1085: Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors

Abstract BACKGROUND: Bone metastasis is a complex process involving reciprocal interplay between cancer and bone cells. Metastatic prostate cancer (PCA) cells secrete extracellular vesicles (EVs), that contain microRNAs (miRNAs), and these can be found in blood. We hypothesize that precursor osteoclasts scavenge PCA EVs, leading to a manipulation of their behavior thereby driving metastasis. METHODS: To test how EV-miRNAs affect metastasis, we stably overexpressed two metastasis-related miRNAs in PCA cells. Bone metastases were induced by inoculating miRNA overexpressing PCA cells into nude mice via intracardiac route with or without the presence of subcutaneous tumors. Tumor progression was monitored by F-luciferase signal (IVIS) while bones were analyzed by microCT ex vivo. miRNAs expression was analyzed by qRT-PCR on precursor osteoclasts isolated from mice blood. Osteoclast differentiation assays were performed using human peripheral mononucleated blood cells on bone chips together with RANKL and M-CSF to drive differentiation. RESULTS: In vivo, intracardiac injection of PC3 cells that overexpress selected miRNAs accelerated the formation of bone metastases (0-85%) compared to control PC3 cells (43%). To determine whether EVs secreted by PCA cells affect osteoclasts, human monocytes were allowed to differentiate to osteoclasts while exposing them to PCA EVs. EVs secreted by control PCA cell lines increased osteoclast differentiation by 22.5% (p<0.01), compared to EVs secreted by RWPE1 noncancerous prostate cells. Exposure to EVs secreted by the miRNA overexpressing PCA cells further enhanced osteoclast formation by an additional 10% (p<0.05). To test whether pro-metastatic miRNAs enhance metastases when mouse osteoclast precursors are pre-stimulated (via EVs or soluble factors), subcutaneous tumours of pro-metastatic cells were grown for 2 weeks. In circulating osteoclast precursors of mice that were pre-stimulated with PCA factors that were secreted by subcutaneous tumors (2 weeks), the miRNA levels were increased by 1.5 - 4 fold (p<0.05), demonstrating that these cells scavenge the secreted tumor miRNAs. Subsequently, mice received an intracardiac injection with matching cells to induce metastases. Trabecular bone volume of >15% was used as indicator for metastasis induced bone loss. Mice that grew both a subcutaneous tumor and also received an intracardiac injection to drive metastasis formation led to an ~30% increased number of mice with >15% bone loss (p<0.01). This indicates that osteoclasts may be manipulated by the cancer cell-secreted factors to form a pro-metastatic niche. CONCLUSION: PCA miRNAs secreted via EVs stimulate osteoclast formation, potentially increasing the formation of overt metastases. Further investigation is needed to unravel novel pathways that can be targeted to prevent bone metastasis. Citation Format: L. Ayse Erozenci, Ning Wang, Albert A. Geldof, Jorn Mulder, Connie R. Jimenez, R. Jeroen van Moorselaar, Allison Gartland, Teun J. de Vries, Irene V. Bijnsdorp. Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors [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 1085.

Abstract 2117: Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis

Abstract Background: Bone metastasis is one of the major clinical concerns that causes skeletal-related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The metastatic prostate cancer cells interact with bone cells (osteoblasts and osteoclasts), resulting in an imbalance in the bone homeostasis leading to increased activation of osteoblasts over osteoclasts. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts (OC) induced by PCa cells and acts as a negative regulator of osteoclasts' function. We hypothesize that PCa-induced NRP2 expression in OC is necessary for low osteolytic activity and thus favors an osteoblastic lesion in PCa bone metastasis. Methods: Mouse OC precursors were isolated from bone marrow of C57BL/6 mice as well as transgenic CSF1R-cre; NRP2 Flox/Flox inducible mice where addition of 4-hydroxytamoxifen depletes NRP2 specifically from the myeloid cells. Differentiation of OCs was conducted under the conditions of RANKL and M-CSF and in conditioned medium (CM) collected from PCa cell line LNCaP C4-2B (promotes high osteoblastic and low osteoclastic activity) and PC3 (predominantly osteoclastic activity) to mimic the conditions in normal bone and PCa bone metastasis. NRP2 expression at protein and mRNA was evaluated. TRAP staining and pit formation were conducted to confirm the differentiation and function of OCs. Results and Discussion: We observed an increase in NRP2 expression in OCs induced by RANKL and M-CSF and in PC3 and LNCaP C4-2B CM simultaneously. TRAP staining and activity confirmed the differentiation of OCs under these conditions. Interestingly, depletion of NRP2 and treatment either in RANKL and M-CSF or LNCaP C4-2B CM exhibited a drastic increase in osteoclast differentiation and function. mRNA analysis revealed an increase in expression of osteoclastic genes following NRP2 depletion in RANKL and M-CSF and LNCaP C4-2B CM. However, NRP2-depleted OC precursors when treated with PC3 CM showed no change in osteoclastogenesis. These findings advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis and that osteolytic PCa evades NRP2 inhibition. Using in vitro and transgenic mice, we will elucidate the molecular mechanisms through which NRP2 regulates osteoclast differentiation and function in normal bone and in LNCaP C4-2B CM. We will also address how PC3 CM-induced OCs escapes the inhibition of NRP2. Together, this approach will elucidate the role of NRP2 axis on OCs in promoting PCa-induced bone metastasis and will aid in determining whether NRP2 axis can be a therapeutic target. Citation Format: Navatha Shree Polavaram, Arup Bag, Samikshan Dutta, Kaustubh Datta. Role of Neuropilin 2 in orchestrating the functions of osteoclasts in promoting prostate cancer bone metastasis [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 2117.

Abstract 5412: miR155 and miR9 expression in human prostate cancer cell lines and prostate cancer tissue

Abstract Background: Differential expression of miRNAs between malignant, precancerous lesions, and nonmalignant prostate tissues may indicate that these molecules play a role in cancer biology. Several miRNAs have been commonly reported to be differentially expressed in prostate cancer, compared to benign prostate tissue, but some results have been contradictory. Expression patterns of specific miRNAs across prostate cancer, precancerous lesions, and benign tissue have not been determined. Design: The expression patterns of 20 miRNAs were determined in the metastatic prostate cancer cells lines LNCaP and PC3, using the nontumorigenic prostate cell line PWR-1E as reference. Differentially expressed miR-9 and miR-155 were subsequently evaluated in epithelial cells obtained by laser capture microdissection from cancerous lesions, high-grade prostatic intraepithelial neoplasia, proliferative inflammatory atrophy, and benign tissue from radical prostatectomy and transurethral prostatectomy tissue specimens, from patients diagnosed with prostate cancer. Total RNA was extracted from the cell lines and microdissected epithelial cells and expression of miR-9 and miR-155 was evaluated by qPCR; miRNA levels in individual samples were normalized to corresponding RNU62 levels. Fold-change values were calculated, and p value < 0.05 was considered statistically significant. The Ethical Review Board of the academic institution approved this study. Results: Differential expression of several miRNAs was found between cell lines. Four miRNAs showed a similar expression profile in the cancer cell lines, two were overexpressed in metastatic cell lines LNCaP and PC3 compared to the nontumorigenic PWR-1E cells (hsa-miR-195, hsa-miR-9), and two were underexpressed (hsa-miR-143, hsa-miR-155). Evaluation of miRNA expression in laser capture microdissected epithelial cells showed a trend towards higher expression of miR-9, and lower expression of miR-155 in microdissected epithelial cells derived from prostate tissue with cancer and proliferative inflammatory atrophy compared to epithelial cells from benign tissue. Conclusions: Our results are in agreement with the description of miR-9 as oncomiRNA. miR-155 was underexpressed in LNCaP and PC3 cells in relation to nontumorigenic PWR-1E cells. Also, this miRNA showed a trend towards underexpression in epithelial cells microdissected from prostate tissue with cancer and proliferative inflammatory atrophy compared to epithelial cells from benign tissue. Further studies of these deregulated miRNAs will help clarify their role in prostate tumorigenesis. Citation Format: Ines Benedetti, Juan Rebollo, Niradiz Reyes. miR155 and miR9 expression in human prostate cancer cell lines and prostate cancer tissue [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 5412.

Abstract 1090: The role of connexin43 in prostate cancer motility

Abstract Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease. Citation Format: Abdulaziz A. Aloliqi. The role of connexin43 in prostate cancer motility [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 1090.

Abstract 5208: Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (PCa) patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced PCa patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behaviors of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte® Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients [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 5208.