Bone Metastatic Prostate Cancer Cells Research Articles

Abstract 161: Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages

Abstract Bone metastasis is a major contributor to the mortality of patients with prostate cancer and results in the development of an immunosuppressive microenvironment in the bone marrow. While advanced age increases the risk of bone metastasis, effects of age-related changes on immune microenvironmental homeostasis are not yet clearly understood. To investigate interactions between cancer cells and the immune microenvironment in elderly individuals, we utilized murine-derived specific bone metastatic prostate cancer cells (RM1-BM), which we had previously isolated from bone metastatic mice and transfected with a luciferase-based reporter gene. We then injected the cells into the left ventricle of both 3‒6 months and 22‒24 months, male, wild-type mice to induce bone metastasis, using age-matched mice injected with PBS as a control. We employed in vivo imaging to compare the metastatic levels in both groups of mice every other day. On day 14, we extracted bone marrow cells for CyTOF analysis and femurs and tibias for Hematoxylin and Eosin staining. Interestingly, our preliminary results demonstrated that healthy aged mice exhibited a lower susceptibility to develop bone metastasis when compared to their younger counterparts. A further investigation of the immune subtypes revealed an increase in the percentage of macrophages in CD45+ immune cells in young mice that developed bone metastasis as opposed to age-matched controls. However, the percentage decreased in aged mice, going from 12.20% to 9.92% (p = 0.001). Lastly, macrophages were isolated from the bone marrow of 3‒6 months and 22‒24 months old mice and were co-cultured with ovalbumin-transfected RM1-BM cells (RM1-BM-ova) in vitro for 24 hours. The flow cytometry results indicated that both groups consist mainly of CD206+ (M2) macrophages. Nonetheless, those obtained from aged mice exhibited reduced ovalbumin presentation capability while maintaining a steady level of type I major histocompatibility complex (MHC-I). These findings suggested that increased infiltration of pro-inflammatory M2 macrophages within the tumor microenvironment may contribute to the bone metastasis of prostate cancer. Therefore, inhibiting M2 macrophages recruitment to the bone marrow microenvironment may prove a useful approach for treating patients with prostate cancer bone metastasis. This study was supported by the NSFC projects (81972420, 81972766, 82173336), and grants from the Shenzhen Science and Technology Innovation Commission (JCYJ20190809161811237, JCYJ20210324104214040). Citation Format: Xiao Zhang, Wei Yu, Xin Huang, Yi Lu, Jian Zhang. Aged mice decelerate bone metastasis of prostate cancer by decreasing infiltration of M2 macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 161.

Abstract 61: PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy

Abstract Recurrent metastatic prostate cancer (PC) typically manifests in the skeleton. Despite our knowledge that DTCs give rise to incurable bone metastatic PC, the molecular mechanisms underpinning their entry into dormancy and exit, remain under-investigated. To address this, we developed a novel in vitro model of PC dormancy as determined by increased entry into G0/G1 state (flowcytometry) and increased p38/ERK activity ratio & p27 (immunoblotting). Interestingly, we also observed dormancy entry resulted in the formation of clusters that remained dormant for long periods (21 days) and could be reawakened via serum addition. RNASeq and bioinformatic analysis of control vs. dormant cells revealed PRDM16 to be significantly elevated during entry into dormancy across mouse (RM1) and human cell lines (LAPC4, 22RV1). In vitro, we observed that genetic silencing of PRDM16 leads to a significant reduction in the ability of PC cells to enter dormancy. Analyses show that PRDM16 increases the expression of anti-apoptotic proteins including BCL-2 while inhibiting pro-apoptotic proteins such as BIM and NOXA. Our preliminary data shows that PRDM16 expression in CRPC cells can be induced by BMP7 and conversely decreased by the BMP signaling antagonist, noggin, factors with known critical roles in regulating bone homeostasis. In vivo, we used intrailiac-artery technique to deliver actively growing RM1 cells or dormant RM1 clusters (D-RM1) to the hind limbs (n = 8) of syngeneic mice (C57BL/6). Bioluminescence shows that D-RM1 remains dormant for more than 30 days. Subsequent histological analyses show that D-RM1 home preferentially to the endosteal niche where, compared to RM1 controls, they remain largely negative for ki-67 and cleaved caspase 3 and exhibit high expression of PRDM16. In conclusion, our data points to PRDM16 as a key regulator of survival pathways during PC entry into dormancy. Citation Format: Mostafa Nasr, Tao Li, Ryan T. Bishop, Conor C. Lynch. PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 61.

The inflammatory macrophages repress the growth of bone metastatic human prostate cancer cells via TNF-α and IL-6 signaling: Involvement of cell signaling regulator regucalcin

Macrophages in the cancer microenvironments may play a regulatory role in the progression and metastasis of prostate cancer cells. However, the crosstalk between macrophages and prostate cancer cells is poorly understood. This study elucidates whether inflammatory macrophages regulate the proliferation and death of human prostate cancer cells in vitro. The RAW264.7 mouse macrophages were cocultured with PC-3 or DU-145 wild-type cells by using a Transwell chamber in vitro. RAW264.7 cells were cocultured with PC-3 or DU-145 cells in the presence of lipopolysaccharide (LPS). This coculturing blocked the proliferation and accelerated the death of cancer cells. Interestingly, cancer cell proliferation was repressed and death was promoted by the addition of the conditioned medium obtained from RAW264.7 cells treated with LPS. Culturing with LPS mostly augmented the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture medium of RAW264.7 cells. The effects of the conditioned medium on the proliferation and death of PC-3 or DU-145 cells were blocked by NF-κB or STAT3 signaling inhibitors. Moreover, the effects of the conditioned medium on the proliferation and death of prostate cancer cells were not expressed in regucalcin-overexpressing cancer cells that diminish the levels of NF-κB p65 and STAT3. Culturing with extracellular TNF-α, IL-6, or regucalcin triggered inhibition of the proliferation of PC-3 wild-type cells. The levels of regucalcin in PC-3 cells were elevated by TNF-α or IL-6 stimulation. This study demonstrates that inflammatory macrophages triggered the loss of prostate cancer cells via the signaling process of NF-κB, STAT3, or regucalcin.

Protein Kinase A (PKA) as a Master Regulator of the Early Metabolic Reprogramming in Bone Metastatic Prostate Cancer Cells

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P < .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

Overexpression of regucalcin blocks the migration, invasion, and bone metastatic activity of human prostate cancer cells: Crosstalk between cancer cells and bone cells.

Prostate cancer is a bone metastatic cancer and is the second leading cause of cancer-related death in men. Prolonged progression-free survival of prostate cancer patients is associated with high regucalcin expression in the tumor tissues. This study investigates the underlying mechanism by which regucalcin prevents bone metastatic activity of prostate cancer cells. Human prostate cancer PC-3 or DU-145 wild-type cells or regucalcin-overexpressing PC-3 or DU-145 cells (transfectants) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Overexpressed regucalcin suppressed the migration and invasion of bone metastatic human prostate cancer cells in vitro, and it reduced the levels of key proteins in metastasis including Ras, Akt, MAPK, RSK-2, mTOR, caveolin-1, and integrin β1. Invasion of prostate cancer cells was promoted by coculturing with preosteoblastic MC3T3-E1 or preosteoclastic RAW264.7 cells. Coculturing with cancer cells and bone cells repressed the growth of preosteoblastic cells and enhanced osteoclastogenesis of preosteoclastic cells, and these alterations were caused by a conditioned medium from cancer cell culture. Disordered differentiation of bone cells was prevented by regucalcin overexpression. Production of tumor necrosis factor-α (TNF-α) in cancer cells was blocked by overexpressed regucalcin. Of note, the effects of conditioned medium on bone cells were prevented by NF-κB inhibitor. TNF-α may be important as a mediator in the crosstalk between cancer cells and bone cells. Overexpression of regucalcin suppressed the migration, invasion, and bone metastatic activity of human prostate cancer cells. This study may provide a new strategy for therapy with the regucalcin gene transfer.

Suppression of bone metastatic castration-resistant prostate cancer cell growth by a suicide gene delivered by JC polyomavirus-like particles.

Prostate cancer is one of the most common cancers in men. The heterogeneity and mutations exhibited by prostate cancer cells often results in the progression to incurable metastatic castration-resistant prostate cancer (mCRPC). Our previous investigations demonstrated that the virus-like particles (VLPs) of JC polyomavirus (JCPyV) can deliver exogenous genes to prostate cancer cells for expression. JCPyV VLPs packaging pPSAtk (PSAtk-VLPs) possess the ability to transcriptionally target and selectively induce cytotoxicity in prostate cancer cells in vitro and in vivo, as pPSAtk can only express the thymidine kinase gene, a suicide gene, in androgen receptor-positive cells. To further investigate whether PSAtk-VLPs inhibit the growth of metastasized prostate cancer cells, we established an animal model of bone-metastatic prostate cancer to compare PSAtk-VLPs with leuprorelin acetate and enzalutamide, hormonal agents commonly used in clinical settings, and investigated the effectiveness of PSAtk-VLPs. In the present study, we observed that PSAtk-VLPs effectively inhibited the growth of prostate cancer cells that had metastasized to the bone in the metastatic animal model. In addition, PSAtk-VLPs showed a higher effectiveness than hormone therapy in this animal model study. These results suggest that PSAtk-VLPs may serve as a treatment option for mCRPC therapy in the future.

Kinetics of radium-223 and its effects on survival, proliferation and DNA damage in lymph-node and bone metastatic prostate cancer cell lines

Background Metastatic castration-resistant prostate cancer (mCRPC) is associated with a very unfavorable prognosis. At this advanced stage of the disease, there are several therapeutic strategies approved in recent times, being one of them Radium-223 dichloride (Radium-223). However, its mechanisms of action and the process that conducts to cell death are not fully understood. Given this, our main goal is to characterize the radiobiological effects induced by Radium-223 and to evaluate its kinetics on metastatic Prostate Cancer (mPCa) cells. Materials and Methods In vitro studies were conducted using two mPCa cell lines, the LNCaP and PC3, the first being derived from lymph node metastasis and the second from bone metastasis. Kinetic studies were conducted to access the capacity of these cell lines to uptake, retain and internalize the Radium-223. For the assessment of radiobiological effects, cells were first exposed to different doses of Radium-223 and the clonogenic assay was done to evaluate cell survival and to determine lethal doses (LD50). Then, the effects were also evaluated in terms of proliferation, oxidative stress, morphological changes and cell damage. Results Radium-223 is uptaken by mPCa cells and reaches the nucleus, where it is retained over time. Irradiation decreases cell survival and proliferation, with LNCaP cells (LD50 = 1.73mGy) being more radiosensitive than PC3 cells (LD50 = 4.20mGy). Irradiated cells showed morphological changes usually associated with apoptosis and a dose-dependent increase in DNA damage. Moreover, activation of cell cycle checkpoints occurs through ATM/CHK2 pathway, which is involved in cell cycle arrest and cell death. Conclusions The cytotoxic and anti-proliferative effects on both cell lines showed that Radium-223 can decrease the aggressiveness of tumor cells by decreasing the cell survival and proliferation and, also, by increasing the DNA damage. The similar results observed in both cell lines indicated that Radium-223 may have the potential to be used as a therapeutic option also for mCRPC patients with lymph node metastasis. The activation of DNA Damage Response pathways allows the possibility to understand the importance of these checkpoints as targets for new combined therapies.

Cleavage of the Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex by Matrix Metalloproteinase 7/Matrilysin Triggers Prostate Cancer Cell Dyscohesion and Migration.

The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell–cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)’s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell–cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.

A NF-ĸB-Activin A signaling axis enhances prostate cancer metastasis.

Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-β family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDHhi CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential.

Betaglycan drives the mesenchymal stromal cell osteogenic program and prostate cancer-induced osteogenesis.

Bone metastatic prostate cancer provokes extensive osteogenesis by driving the recruitment and osteoblastic differentiation of mesenchymal stromal cells (MSCs). The resulting lesions greatly contribute to patient morbidity and mortality, underscoring the need for defining how prostate metastases subvert the MSC-osteoblast differentiation program. To gain insights into this process we profiled the effects of co-culture of primary MSCs with validated bone metastatic prostate cancer cell line models. These analyses revealed a cast of shared differentially induced genes in MSC, including betaglycan, a co-receptor for TGFβ. Betaglycan has not been studied in the context of bone metastatic disease previously. Here we report that loss of betaglycan in MSC is sufficient to augment TGFβ signaling, proliferation and migration, and completely blocks the MSC-osteoblast differentiation program. Further, betaglycan was revealed as necessary for prostate cancer-induced osteogenesis in vivo. Mechanistically, gene expression analysis revealed betaglycan controls the expression of a large repertoire of genes in MSCs, and that betaglycan loss provokes >60-fold increase in the expression of Wnt5a that plays important roles in stemness. In accord with the increased Wnt5a levels, there was a marked induction of canonical Wnt signaling in betaglycan ablated MSCs, and the addition of recombinant Wnt5a to MSCs was sufficient to impair osteogenic differentiation. Finally, the addition of Wnt5a neutralizing antibody was sufficient to induce the expression of osteogenic genes in betaglycan-ablated MSCs. Collectively, these findings suggest a betaglycan-Wnt5a circuit represents an attractive vulnerability to ameliorate prostate cancer-induced osteogenesis.

Abstract 1992: Mesenchymal stem cell-derived interleukin-28 can drive the selection of apoptosis resistant bone metastatic prostate cancer

Abstract Bone metastatic prostate cancer is painful and incurable. We believe that understanding the interactions of tumor cells and the surrounding stromal cells may provide new therapeutic options. Bone metastatic prostate cancer promotes mesenchymal stem cell (MSC) recruitment and differentiation into osteoblasts, leading to the clinically often-observed osteogenic phenotype. However, the reciprocal roles of bone- marrow derived MSCs on prostate cancer cells are less explored. Here, we discovered that MSCs significantly suppress prostate cancer growth in vitro and in vivo. Mechanistically, we show that MSC-derived interleukin-28 (IL-28) promotes prostate cancer cell apoptosis via the IL-28 receptor alpha (IL-28Rα). However, chronic exposure to MSCs yields prostate cancer cell populations that are resistant to IL-28 induced apoptosis and therapeutics such as docetaxel. In vivo, we observed that MSC selected prostate cancer cells rather than being suppressed, grow at a significantly enhanced rate in bone. The increase in apoptosis resistance is accompanied by a shift in downstream signaling of the IL-28Rα from STAT1 to STAT3. Reduction of STAT3 levels or pharmacological inhibition (STI-201) significantly reduces the growth of MSC selected prostate cancer cells in vitro and in vivo. Collectively, these data shed light on how MSCs in the bone marrow microenvironment initially protect against prostate cancer cell establishment but in doing so, help select for aggressive, apoptosis resistant bone metastatic prostate cancer cells that are more aggressive. Given that the majority of prostate to bone metastases are positive for STAT3 expression and activity, our data provide rationale for the therapeutic targeting of STAT3 in bone metastatic prostate cancer. Citation Format: Jeremy J. Mcguire, Leah Cook, Jeremy Frieling, Ayaz Muhammad, Harshani Lawrence, Nicholas Lawrence, Conor Lynch. Mesenchymal stem cell-derived interleukin-28 can drive the selection of apoptosis resistant bone metastatic 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 1992.

Abstract 1913: Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment

Abstract Cancer is a heterogeneous collection of neoplastic cells, surrounded by non-malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross-talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can inhibit cancer growth and proliferation during the initial stages of carcinogenesis; however, cancer cells can evade these inhibitory signals, and reshape the TME, making it more amenable to their own growth. This gives rise to cancer-associated stromal cells, including cancer-associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial-mesenchymal transition. Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan (HSPG) that has both pro-mitogenic and pro-metastatic activity. GPC1’s role in regulating fibroblasts and their transformation into CAFs has not been investigated. We studied the ability of prostate cancer cells to alter the TME in vitro by exposing human bone fibroblasts (HS-5 cells) to tumor conditioned media (TCM) isolated from a human neuroendocrine (DU-145) and bone (PC-3) metastatic prostate cancer cell lines and assessed changes in cell morphology and cytotoxicity (using MTT). Exposure of HS-5 cells to TCM from DU-145 and PC-3 cells altered cellular morphology to a more mesenchymal phenotype similar to that seen in active myofibroblasts. TCM also decreased MTT staining in a time-dependent manner. We also determined the effect of GPC1 knockdown (KD) in HS-5 cells on cell morphology, migration and cytotoxicity. Interestingly, we saw phenotypic switching of GPC1 KD HS-5 cells to a myofibroblast like morphology akin to that when treated with TCM. There was no change in cytotoxicity in GPC1 KD HS-5 cells as compared to control cells. However, GPC1 KD enhanced the migration of HS-5 cells in wound healing assay. Migration was also increased in GPC1 KD HS-5 cells upon treatment with TCM. GPC1 KD also increased HS-5 cell contractility significantly. Collectively, these data support the hypothesis that GPC1 plays an inhibitory role in the TME of prostate cancer by regulating the activation of fibroblasts. Citation Format: Sukhneeraj P. Kaur, Hee K. Lee, Robert D. Arnold, Brian S. Cummings. Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment [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 1913.

Abstract 2960: NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to be beneficial in preventing various types of cancers including prostate cancer (PCa). Of all the NSAIDs in use, Aspirin appears to be one of the promising anticancer agents for PCa management. However, experimental and epidemiological studies indicated that larger doses of Aspirin and long term supplementation is required to achieve anticancer effect with accompanied side effects. Therefore, our study focused on evaluating Aspirin derivative, namely NCX4040 which is a NO releasing form of Aspirin as a potent alternative for PCa treatment while minimizing the side effects. In the present study, we assessed the anticancer effects of NCX4040 on PC-3, a bone metastatic PCa cell line employing various in-vitro cellular assays. In our preliminary studies, we compared the effects of NCX4040 with that of Aspirin and NO releasing compound DETA/NO on PC-3 cells using MTT as read out. These results showed that NCX4040 is highly potent in inhibiting the viability of PC-3 cells when compared to Aspirin or DETA/NO treatments. Hence, further studies were focused on characterizing the anticancer properties as well as the underlying anticancer mechanism of NCX4040. Of note, our results showed that NCX4040 robustly inhibited the colony forming ability of PC-3 cells in-vitro implicating its antitumorigenic potential. In addition to inhibiting colony formation of PC-3 cells, NCX4040 also suppressed the Transwell matrigel invasion of PC-3 cells suggesting the antimetastatic effects of NCX4040. The observed antimetastatic effect of NCX4040 in PC-3 PCa cells appears to be via targeting actin cytoskeleton as confirmed by confocal microscopic analysis using phalloidin stain. Following these findings, we performed cell cycle analysis to determine whether NCX4040 enforces cell cycle arrest in PC-3 cells. These results showed that NCX4040 induced robust G0 cell cycle arrest in a dose dependent manner. Moreover, flow cytometry analysis using Annexin V and PI staining revealed that NCX4040 induced potent apoptotic cell death in PC-3 cells. To delineate the underlying anticancer mechanism of NCX4040, PC-3 cells treated with NCX4040 was subjected to apoptotic protein array analysis. Results of this study inferred that NCX4040 down regulated the expression of key anti-apoptotic proteins such as Survivin, XIAP and IAPs. In conclusion, our study suggests that NCX4040 is a promising alternative agent for PCa treatment. Citation Format: SOMAIAH CHINNAPAKA, Gnanasekar Munirathinam. NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for 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 2960.

Parallel-Reaction-Monitoring-Based Proteome-Wide Profiling of Differential Kinase Protein Expression during Prostate Cancer Metastasis in Vitro.

Prostate cancer is the most common type of cancer in men, and kinases are heavily pursued as drug targets for anticancer therapy. In this study, we applied our recently reported parallel-reaction-monitoring (PRM)-based targeted proteomic method to examine the reprogramming of the human kinome associated with bone metastasis of prostate cancer in vitro. The method displayed superior sensitivity over the shotgun-proteomic approach, and it facilitated the quantification of the relative expression of 276 kinase proteins in a pair of bone metastatic prostate cancer cells. Among the differentially expressed kinases, mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) stimulates the migration and invasion of cultured prostate cancer cells, partially by modulating the activity of secreted matrix metalloproteinases 9 (MMP-9). We also found that the upregulation of MAP4K4 in metastatic prostate cancer cells is driven by the MYC proto-oncogene. Cumulatively, we identify MAP4K4 as a potential promoter for prostate cancer metastasis in vitro.

Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis

The role of extracellular vesicles (EVs) as vehicles for cell-to-cell communication between a tumour and its environment is a relatively new concept. The hypothesis that EVs may be critical in co-opting tissues by tumours to generate distant metastatic niches is particularly pertinent to prostate cancer (PCa), where metastatic-tropism to bone predominates over other tissue types. The potential role of EVs as a means of communication between PCa cells and cells of the bone stroma such as osteoblasts, is yet to be fully explored. In this study, we demonstrate that PCa cell EVs both enhance osteoblast viability and produce a significantly more supportive growth environment for PCa cells when grown in co-culture with EV-treated osteoblasts (p < 0.005). Characterisation of the RNA cargo of EVs produced by the bone-metastatic PCa cell line PC3, highlights the EV-RNA cargo is significantly enriched in genes relating to cell surface signalling, cell–cell interaction, and protein translation (p < 0.01). Using novel techniques to track RNA, we demonstrate the delivery of a set of PCa-RNAs to osteoblast via PCa-EVs and show the effect on osteoblast endogenous transcript abundance. Taken together, by using proof-of-concept studies we demonstrate for the first time the contribution of the RNA element of the PCa EV cargo, providing evidence to support PCa EV communication via RNA molecules as a potential novel route to mediate bone metastasis. We propose targeting PCa EVs could offer a potentially important preventative therapy for men at risk of metastatic PCa.

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.

Abstract 4465: Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells

Abstract Glypican-1 (GPC-1) is a cell surface heparan sulfate proteoglycan that mediates cell proliferation, differentiation and migration. It also possess oncogenic functions in breast, pancreatic and lung cancer cells, and is a proposed biomarker for prostate cancer. Despite these data the function of GPC-1 in prostate cancer is not fully known. We addressed this gap-in-knowledge using in vitro and in vivo mouse models. We first showed that GPC-1 was overexpressed in bone metastatic prostate cancer cells (PC-3) but was not detected in lymph node metastatic cancer cells (LNCaP) and non-cancerous prostate cells (RWPE-1). Next, we inhibited GPC-1 expression using shRNA in bone metastatic cancer cells (PC-3), and verified inhibition using western blot analysis and qRT-PCR. Crystal violet staining, trans-well assays, matrigel colony cultures and adhesion assays were used to determine the effect of GPC-1 inhibition on cancer cell growth, migration, spheroid formation and cell adhesion. Inhibition of GPC-1 reduced prostate cancer cell growth, migration and spheroid formation; however, there were no changes in cell adhesion. These in vitro data suggested that GPC-1 played a role in prostate cancer progression by acting as an oncogene. We further investigated this possibility in vivo using athymic (NCr) mice xenografts. Interestingly, inhibition of GPC-1 in PC-3 cells appeared to increase tumor size as compared to scrambled controls. Further analysis showed that that inhibition of GPC-1 increased the tumor expression of MMP-9 and N-cadherin, but not E-cadherin. We hypothesized that the discrepancy between the in vitro and in vivo results may be mediated by interaction of PC-3 cells with the tumor microenvironment. We addressed this hypothesis by testing the effect of tumor conditioned media (TCM) from control and GPC-1 knockdown cells on human bone marrow derived mesenchymal stem cells (hMSC) and fibroblast (HS-27). Treatment of both hMSC and HS-27 cells with TCM from GPC-1 knockdown PC-3 cells increased MMP-9 and interestingly MMP-2 expression and activity as compared to control cells. We also detected increases in the expression of N-Cadherin in a co-culture model. Collectively, these data suggest that GPC-1 acts as an oncogene to enhance prostate cancer cell growth in vitro; however, these data also suggest the GPC-1 may act as a tumor suppressor in vivo. This discrepancy may be due to the interaction off GPC-1 with the tumor microenvironment, which would not be detected in single-cell culture models. Citation Format: Nhat D. Quach, Matthew Eggert, Deepraj Ghosh, Michelle R. Dawson, Robert Arnold, Brian S. Cummings. Glypican-1: A tumor suppressor or an oncogene in human bone metastatic prostate cancer cells [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 4465. doi:10.1158/1538-7445.AM2017-4465

\u0394Np63\u03b1 promotes adhesion of metastatic prostate cancer cells to the bone through regulation of CD82

ΔNp63α is a critical mediator of epithelial development and stem cell function in a variety of tissues including the skin and breast, while overexpression of ΔNp63α acts as an oncogene to drive tumor formation and cancer stem cell properties in squamous cell carcinoma. However, with regards to the prostate, while ΔNp63α is expressed in the basal stem cells of the mature gland, during adenocarcinoma development, its expression is lost and its absence is used to clinically diagnose the malignant state. Surprisingly, here we identify a sub-population of bone metastatic prostate cancer cells in the PC3 cell line that express ΔNp63α. Interestingly, we discovered that ΔNp63α favors adhesion and stem-like growth of these cells in the bone microenvironment. In addition, we show that these properties require expression of the target gene CD82. Together, this work uncovers a population of bone metastatic prostate cancer cells that express ΔNp63α, and provides important information about the mechanisms of bone metastatic colonization. Finally, we identify metastasis-promoting properties for the tetraspanin family member CD82.

Keratin 13 expression reprograms bone and brain metastases of human prostate cancer cells.

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases.

Abstract 749: Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment

Abstract Prostate cancer frequently metastasizes to bone, resulting in increased risk of fractures and severe pain that significantly impacts patient quality of life. Bone metastatic prostate cancer is currently incurable with standard of care therapies being mainly palliative. In bone, prostate cancer generates extensive osteogenic lesions by promoting osteoblast activity. Osteoblasts are specialized bone forming cells derived from mesenchymal stem cells. In a bid to find new molecular targets through which prostate cancer induces osteogenesis, we incubated naïve MSCs with osteogenic bone metastatic prostate cancer cell line C4-2B conditioned media. Gene expression analysis revealed a 3-fold increase in expression of the TGFâ co-receptor, betaglycan. Given the critical role of TGFâ in the context of bone metastatic disease, we examined the role of betaglycan in MSC behavior. To this end, we utilized shRNA to knockdown betaglycan gene expression in mouse bone marrow-derived primary MSCs. Reduced betaglycan expression in knockdown (KD) MSCs had little to no impact on the expression of TGFâ signaling receptors, TGFâRI and TGFâRII, or pan-TGFâ ligand expression. However, using PAI promoter activity, a readout for TGFâ signaling, we observed a 3-fold increase in luminescence in KD-MSCs compared to controls. Likewise, phosphorylated Smad 2 was significantly increased in Betaglycan KD-MSCs in response to TGFâ treatment, collectively demonstrating increased TGFâ signaling in MSCs with reduced betaglycan expression. Using modified Boyden chamber assays, we found that 50% more Betaglycan-KD MSCs migrated towards recombinant TGFâ than controls (100 KD-MSCs cells migrated towards recombinant TGFâ vs. 50 control cells); this change was reversed with the addition of recombinant betaglycan. Using osteogenic differentiation assay, we found that Betaglycan KD-MSC differentiation was reduced by approximately 70% compared to control MSCs as measured by alizarin red staining. Addition of recombinant TGFâ further suppressed differentiation in both KD-MSCs and Control MSCs. This phenomenon was rescued by the addition of recombinant betaglycan, suggesting that MSC-derived betaglycan is critical for driving the osteogenic program. We are currently determining the impact of betaglycan on MSC behavior in the tumor bone microenvironment using in vivo models of bone metastatic prostate cancer. Collectively, these findings suggest that prostate cancer-induced MSC osteogenic programs are regulated in part via the induction of betaglycan. Citation Format: Leah M. Cook, Jeremy J. McGuire, Conor C. Lynch. Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment. [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 749.