Metastatic Prostate Cancer Cells Research Articles

Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype

Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR.

Engineering A11 Minibody-Conjugated, Polypeptide-Based Gold Nanoshells for Prostate Stem Cell Antigen (PSCA)-Targeted Photothermal Therapy.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

The possible association of intracellular Ca2+ with metastasis in human cancer cells is poorly understood. We have studied Ca2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca2+ was measured using a membrane-permeant fluorescent Ca2+-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca2+ level could be induced by applying a high-K+ solution. Various parameters of the oscillations depended on extracellular Ca2+ and voltage-gated Na+ channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na+ channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca2+ oscillations. It is concluded that the functional voltage-gated Na+ channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca2+ activity. Possible mechanisms and consequences of the Ca2+ oscillations are discussed.

Global and Targeted Proteomics of Prostate Cancer Cell Secretome: Combination of 2-Dimensional Image-Converted Analysis of Liquid Chromatography and Mass Spectrometry and In Silico Selection Selected Reaction Monitoring Analysis

Prostate-specific antigen is currently the only protein biomarker routinely used as a diagnostic tool for early detection and treatment monitoring of prostate cancer. However, it remains questionable whether prostate-specific antigen-based screening can sensitively and selectively identify the presence and progression status of primary and metastatic prostate cancers. Hence, the purpose of this study was to identify potential biomarker candidates in the secretome of primary and metastatic prostate cancer cells by using a combination of global and targeted proteomics. Quantitative comparisons among secretome proteins derived from androgen-responsive primary cancer cells (P-22Rv1), androgen-irresponsive bone metastatic cancer cells (M-PC-3), and noncancerous prostate cells (N-PNT2) were performed using 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry followed by in silico selection selected reaction monitoring analysis. Mediator of RNA polymerase II transcription subunit 13-like, insulin-like growth factor-binding protein 2, and hepatocyte growth factor were identified as highly secreted proteins from P-22Rv1 cells compared with N-PNT2 cells. Prostate-associated microseminoprotein, proactivator polypeptide, collagen-α-1 (VI) chain, and neuropilin-1 were identified as predominantly secreted proteins in M-PC-3 cells compared with N-PNT2 cells. These proteins in biological fluids are considered to be candidate biomarkers of primary and/or metastatic prostate cancer.

Bone marrow adipocytes promote the Warburg phenotype in metastatic prostate tumors via HIF-1α activation.

Metabolic adaptation is increasingly recognized as a key factor in tumor progression, yet its involvement in metastatic bone disease is not understood. Bone is as an adipocyte-rich organ, and a major site of metastasis from prostate cancer. Bone marrow adipocytes are metabolically active cells capable of shaping tumor metabolism via lipolysis and lipid transfer. In this study, using in vitro and in vivo models of marrow adiposity, we demonstrate that marrow fat cells promote Warburg phenotype in metastatic prostate cancer cells. We show increased expression of glycolytic enzymes, increased lactate production, and decreased mitochondrial oxidative phosphorylation in tumor cells exposed to adipocytes that require paracrine signaling between the two cell types. We also reveal that prostate cancer cells are capable of inducing adipocyte lipolysis as a postulated mechanism of sustenance. We provide evidence that adipocytes drive metabolic reprogramming of tumor cells via oxygen-independent mechanism of HIF-1α activation that can be reversed by HIF-1α downregulation. Importantly, we also demonstrate that the observed metabolic signature in tumor cells exposed to adipocytes mimics the expression patterns seen in patients with metastatic disease. Together, our data provide evidence for a functional relationship between marrow adipocytes and tumor cells in bone that has likely implications for tumor growth and survival within the metastatic niche.

Molecular Markers and Targeted Therapeutics in Metastatic Tumors of the Spine: Changing the Treatment Paradigms.

A review of the literature. The aim of this study was to discuss the evolution of molecular signatures and the history and development of targeted therapeutics in metastatic tumor types affecting the spinal column. Molecular characterization of metastatic spine tumors is expected to usher in a revolution in diagnostic and treatment paradigms. Molecular characterization will provide critical information that can be used for initial diagnosis, prognosticating the ideal treatment strategy, assessment of treatment efficacy, surveillance and monitoring recurrence, and predicting complications, clinical outcome, and overall survival in patients diagnosed with metastatic cancers to the spinal column. A review of the literature was performed focusing on illustrative examples of the role that molecular-based therapeutics have played in clinical outcomes for patients diagnosed with metastatic tumor types affecting the spinal column. The impact of molecular therapeutics including receptor tyrosine kinases and immune checkpoint inhibitors and the ability of molecular signatures to provide prognostic information are discussed in metastatic breast cancer, lung cancer, prostate cancer, melanoma, and renal cell cancer affecting the spinal column. For the providers who will ultimately counsel patients diagnosed with metastases to the spinal column, molecular advancements will radically alter the management/surgical paradigms utilized. Ultimately, the translation of these molecular advancements into routine clinical care will greatly improve the quality and quantity of life for patients diagnosed with spinal malignancies and provide better overall outcomes and counseling for treating physicians. N/A.

Comprehensive Analysis of Secreted Protein, Acidic and Rich in Cysteine in Prostate Carcinogenesis: Development of a 3D Nanostructured Bone-Like Model.

Most aggressive prostate cancer (PCa) types tend to metastasize frequently to bone and SPARC, a matricellular protein, might participate in such biological processes. The objective of this study was to evaluate the effect of SPARC in prostate carcinogenesis and bone metastization. This was explored assessing the morphology, metabolic activity and SPARC expression of different PCa cell lines resembling different stages of carcinogenesis, using a 3D bone-biomimetic model (collagen nanofibers/nanohydroxyapatite) grafted with SPARC. Our findings highlight distinct cellular behavior depending on cell type and presence of exogenous SPARC. In fact, SPARC addition contributed to the survival and significant growth of a non-bone metastatic PCa cell line (LNCaP) on bone-like biomaterial. Moreover, SPARC expression levels were evaluated in a series of prostatic tissues, comparing normal prostate, pre-neoplastic prostate intraepithelial neoplasias and overtly malignant tumors, and also metastasis to its correspondent primary prostate tumors, ascertaining potential association between SPARC and clinicopathological data. Remarkably, SPARC was overexpressed in patients with higher Gleason Score, indicating tumors with poor prognosis, as well as in metastasis, particularly from bone sites, compared with their respective primary tumors. The results suggest a potential role of SPARC as a clinical target on PCa, due to its association with bone metastization.

Abstract 1630: Dysregulation of Hippo pathway in metastatic prostate cancer

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

Abstract LB-315: Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a

Abstract Bone is a preferential site of metastasis from prostate cancer (PCa). Age and obesity, conditions that increase adipocyte numbers in bone marrow, are risk factors for skeletal metastases from PCa. Research in our laboratory focuses on understanding the interactions between adipocytes and tumor cells that have infiltrated the bone marrow. Specifically, we are examining how the secretion, transport, and uptake of adipocyte-supplied factors promote metastatic progression in bone. We have previously shown that bone marrow adipocytes enhance a glycolytic phenotype in PCa cells through paracrine activation of glycolytic enzymes GLUT1, HK2, PDK1, ENO2, and LDHa at both the mRNA and protein levels. This correlated with an increase in glycolytic activity as measured by higher levels of lactate secretion and decreases in oxidative phosphorylation, indicative of an enhanced Warburg-like metabolic signature. We then assessed HIF-1a activity through mRNA analysis of HIF-1a target genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF). We found that CA9 and VEGF had elevated expression levels in tumor cells exposed to adipocytes, indicative of enhanced HIF-1a activity. Notably, PCa cells exposed to bone marrow adipocytes had significantly enhanced presence of HIF-1a in the nucleus, result further confirming HIF-1α, activation in tumor cells exposed to marrow adipocytes. Hypoxia chamber experiments revealed that these effects were observable regardless of the presence or absence of oxygen, indicating the presence of oxygen-independent HIF-1a signaling or a state of pseudohypoxia in tumor cells. These results were further validated in multiple in vitro cell culture and in vivo mouse models, where an increased production of both glycolytic genes and HIF-1α target genes was shown to correlate with marrow adiposity. Our overall hypothesis is that bone marrow adipocyte-supplied lipids within the bone microenvironment cause HIF-1a activation and metabolic switch to glycolysis in metastatic PCa cells in bone, leading to their increased aggressiveness and resistance to therapy. Citation Format: Jonathan Diedrich, Erandi Rajagurubandara, Mackenzie Herroon, Izabela Podgorski. Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a. [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 LB-315.

Abstract 1171: LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop

Abstract Increased activation of the GPCR CXCR4/CXCL12 signaling axis is commonly noted in metastatic prostate cancer and shows an association with poor prognosis. CXCR4, and its ligand, CXCL12/SDF-1a, are recognized as powerful mediators for tumor microenvironment remodeling and chemoprotection. LIM kinase 1 (LIMK1), a serine/threonine kinase, is an actin and microtubule modulatory protein that is also overexpressed in a variety of cancers including prostate cancer. Earlier, we showed that LIMK1 is involved in cell invasion and inhibition of LIMK1 expression reverted the invasive phenotype of PC3 prostate cancer cells. Activation of CXCR4/CXCL12 axis promotes activation of LIMK1 through RhoGTPAse pathway and promotes docetaxel resistance. Here we show that LIMK1 expression and functions have a positive correlation with CXCR4 expression and subcellular localization. We used metastatic prostate cancer cell lines PC3 and M12 and the non-tumorigenic prostate cell lines BPH and P69 to monitor expression and membrane localization of CXCR4 using western blots, flow cytometry and immunofluorescence analysis. Our analysis showed that cells with higher expression of LIMK1 (PC3) overexpress CXCR4, and that cell lines with low levels of LIMK1 (BPH and P69) express relatively lower amounts of CXCR4. Flow cytometric analysis showed a stronger surface staining of CXCR4 in the LIMK1 overexpressing PC3 cells compared to BPH cells. Next, we studied the effect of shRNA-mediated inhibition of LIMK1 expression on CXCR4 expression in PC3 or M12 cells. Western blot and immunofluorescence analysis showed noticeable reduction in CXCR4 levels, and its surface localization, as noted by flow cytometry, in LIMK1 shRNA transfected PC3 and M12 cells. To assess if the kinase activity of LIMK1 is essential for CXCR4 expression and surface localization, we treated these cells with LIMK1 specific kinase inhibitor BMS-5 and determined expression of CXCR4. Western blots and immunofluorescence analysis showed reduced expression of CXCR4 in treated cells compared to vehicle treated cells. Our observation indicates an indirect association between LIMK1 and CXCR4 expression and shows for the first time that expression of LIMK1 is required for the maintenance of CXCR4 expression. Our study demonstrates a novel positive feed back regulatory mechanism between LIMK1 and CXCR4 and suggest a possible combination treatment option through inhibition of LIMK1 expression and/or its kinase activity for improving chemosensitivity of prostate cancer cells with overactive CXCR4/CXCL12 axis. Citation Format: Richard Ottman, Lisa Ritchey, Ryan Herchan, Alexia Bossan, Ratna Chakrabarti. LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop. [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 1171.

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.

Abstract 243: Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells

Abstract Advanced prostate cancer (PC) is treated primarily by means of chemotherapy with one of the clinically approved microtubule (MT)-targeting agents (MTAs): paclitaxel, docetaxel and cabazitaxel. The mechanism of action of the MTAs on the cellular level is to bind to MTs and inhibit MT-dependent intracellular pathways, ultimately leading to cell death. In PC, MTAs are the only chemotherapy class shown to improve survival, however, there is high variability of response to MTA chemotherapy among patients as well as emergence of resistance to drug action which hampers the clinical efficacy. Clinicians need additional information to match individual patient tumors to the most effective drugs or refrain from treating patients that will not respond. However, the precise mechanism of action or resistance development to MTAs is not understood, which represents a critical gap in our knowledge to select the best treatment. Our hypothesis is that there are inherent differences in tumor MT dynamics among individual patients which dictates their response to treatment with specific MTAs. To this end, we envision that a systematic characterization of PC MT dynamics and their response to MTAs will allow chemotherapy customization and prolong survival. Specifically, our approach consists of the analysis of metastatic castrate resistant PC (mCRPC) organoids (Gao D. et al, 2014) derived from bone (acetabulum, vertebrae), soft tissue (salvage prostatectomy, lymph nodes), pleural effusion and circulating tumor cells biopsy samples obtained from metastatic patients who have received hormonal therapy and the MTA docetaxel or hormonal therapy alone or no treatment. We apply a quantitative single-cell image analysis algorithm (ClusterTrack) to measure a panel of twelve distinct MT dynamics parameters (a MT dynamics signature). Preliminary analysis suggests differences between different mCRPC organoids. MT tracking results indicate a correlation of decreased MT growth rate with increased Op18/stathmin expression. In this context, we hypothesize that differential expression of MT regulating genes affects specific parameters of MT dynamics, thus determining the sensitivity to MTA treatment. Citation Format: Alexandre Matov, Johan de Rooij, Jay Gatlin, Julia Rohrberg, Nikta Ahmad, Rahul Aggarwal, Jeff Simko, Andrei Goga, Charles Ryan, Torsten Wittmann. Investigating microtubule growth dynamics in patient-derived metastatic prostate cancer cells. [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 243.

Abstract 1600: Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling

Abstract Polo-like kinase 1 (PLK1) is a key cell cycle regulator that has been implicated in the development of various cancers, including prostate cancer. However, the functions of PLK1 beyond cell cycle regulation remain poorly characterized. Here, we report that PLK1 overexpression in prostate epithelial cells triggers oncogenic transformation. It also results in dramatic transcriptional reprogramming of the cells, leading to epithelial-to-mesenchymal transition (EMT) and stimulation of cell migration and invasion. Consistently, PLK1 downregulation in metastatic prostate cancer cells enhances epithelial characteristics and inhibits cell motility. The signaling mechanisms underlying the observed cellular effects of PLK1 involve direct PLK1-dependent regulation of CRAF with subsequent stimulation of the MEK1/2-ERK1/2-Fra1-ZEB1/2 signaling pathway. Our findings highlight novel non-canonical functions of PLK1 as a key regulator of EMT and cell motility in normal prostate epithelium and prostate cancer. This study also uncovers a previously unanticipated role of PLK1 as a potent activator of MAPK signaling. Citation Format: Jianguo Wu, Andrei I. Ivanov, Zheng Fu. Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling. [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 1600.

Abstract 1564: Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation

Abstract The objective of this study is to assess the regulatory roles of reactive endosteum associated with foci of bone metastatic prostate cancer cells. Previous studies evaluated tissue arrays of human prostate cancer bone metastases and we identified a “reactive endosteum” spatially associated with foci of prostate cancer cells on trabecular bone. Reactive endosteum was characterized by elevated expression of tenascin-C, a glycoprotein deposited in the extracellular matrix of reactive stroma at sites of wound repair and in the primary tumor microenvironment in adult tissues. In general, tenascin-C is stromal derived in adult tissues. Of interest, tenascin-C is also deposited at sites of fracture repair and at sites of Brodie abscess osteomyelitis-associated inflammation in human bone. Tenascin-C has many diverse functions: it regulates cell adhesion, migration and proliferation in different pathological states while playing an important role in neuropatterning and osteogenesis during development. Moreover, tenascin-C has been shown to regulate several signal transduction pathways. To evaluate prostate cancer-bone interactions, we developed an in vitro, 3D, osteogenic organoid model composed of human mesenchymal stem cells induced to osteogenesis that were combined with human prostate VCaP cells in organ culture. In this model, foci of VCaP cells associated preferentially to regions of high tenascin-C deposition. VCaP cells also preferentially bound to purified human tenascin-C deposited on culture plates in a dose-dependent manner. VCaP cells also exhibited an elevated growth rate on osteo-mimetic plates coated with human tenascin-C as compared to control. Moreover, VCaP cells preferentially bound to human tenascin-C coated bovine trabecular bone cubes in vitro and initiated colony formation. Evaluation of potential mediators identified integrin alpha 9 beta 1 as the key mediator of attachment to human tenascin-C. Neutralization of this integrin inhibited adhesion. Evaluation of potential signaling pathways have implicated activation of EGF receptor (EGFR), Wnk1, and STAT6 pathways. Human tenascin-C exhibits EGF-like repeats and fibronectin type III domains that may mediate activation of these pathways. Of interest, adhesion and growth of VCaP cells on tenascin-C also induced elevated tenascin-C expression in these cells, a novel finding. Additional 3D organoid and in vivo xenograft studies with other cell types support the finding of induced expression of tenascin-C in epithelial cells associated with reactive stroma or matrix. In summary, our studies characterize elevated tenascin-C at sites of a reactive endosteum associated with metastatic prostate cancer foci. Data suggests that tenascin-C mediates adhesion and proliferation of cancer cells via activation of several pathways. These data may aid in developing novel therapeutic approaches to treat metastatic disease. Citation Format: Rebeca San Martin, Kenneth Pienta, David R. Rowley. Reactive endosteum in prostate cancer bone metastases: role of tenascin-C in regulating cancer cell adhesion and proliferation. [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 1564.

New 3D-Culture Approaches to Study Interactions of Bone Marrow Adipocytes with Metastatic Prostate Cancer Cells.

Adipocytes are a major component of the bone marrow that can critically affect metastatic progression in bone. Understanding how the marrow fat cells influence growth, behavior, and survival of tumor cells requires utilization of in vitro cell systems that can closely mimic the physiological microenvironment. Herein, we present two new three-dimensional (3D) culture approaches to study adipocyte–tumor cell interactions in vitro. The first is a transwell-based system composed of the marrow-derived adipocytes in 3D collagen I gels and reconstituted basement membrane-overlayed prostate tumor cell spheroids. Tumor cells cultured under these 3D conditions are continuously exposed to adipocyte-derived factors, and their response can be evaluated by morphological and immunohistochemical analyses. We show via immunofluorescence analysis of metabolism-associated proteins that under 3D conditions tumor cells have significantly different metabolic response to adipocytes than tumor cells grown in 2D culture. We also demonstrate that this model allows for incorporation of other cell types, such as bone marrow macrophages, and utilization of dye-quenched collagen substrates for examination of proteolysis-driven responses to adipocyte- and macrophage-derived factors. Our second 3D culture system is designed to study tumor cell invasion toward the adipocytes and the consequent interaction between the two cell types. In this model, marrow adipocytes are separated from the fluorescently labeled tumor cells by a layer of collagen I. At designated time points, adipocytes are stained with BODIPY and confocal z-stacks are taken through the depth of the entire culture to determine the distance traveled between the two cell types over time. We demonstrate that this system can be utilized to study effects of candidate factors on tumor invasion toward the adipocytes. We also show that immunohistochemical analyses can be performed to evaluate the impact of direct interaction of prostate tumor cells with adipocytes. Our models underline the importance of using the appropriate culture conditions to mimic physiological interactions between marrow adipocytes and metastatic tumor cells. These systems have a potential to be utilized for analyses of various factors that may be regulated by the adipocytes in bone. Their application likely extends beyond metastatic prostate cancer to other tumors that colonize the bone marrow microenvironment.

Controlled Release of Nor-β-lapachone by PLGA Microparticles: A Strategy for Improving Cytotoxicity against Prostate Cancer Cells.

Prostate cancer is one of the most common malignant tumors in males and it has become a major worldwide public health problem. This study characterizes the encapsulation of Nor-β-lapachone (NβL) in poly(d,l-lactide-co-glycolide) (PLGA) microcapsules and evaluates the cytotoxicity of the resulting drug-loaded system against metastatic prostate cancer cells. The microcapsules presented appropriate morphological features and the presence of drug molecules in the microcapsules was confirmed by different methods. Spherical microcapsules with a size range of 1.03 ± 0.46 μm were produced with an encapsulation efficiency of approximately 19%. Classical molecular dynamics calculations provided an estimate of the typical adsorption energies of NβL on PLGA. Finally, the cytotoxic activity of NβL against PC3M human prostate cancer cells was demonstrated to be significantly enhanced when delivered by PLGA microcapsules in comparison with the free drug.

SFMBT2 (Scm-like with four mbt domains 2) negatively regulates cell migration and invasion in prostate cancer cells.

Metastatic prostate cancer is the leading cause of morbidity and mortality in men. In this study, we found that expression level of SFMBT2 is altered during prostate cancer progression and has been associated with the migration and invasion of prostate cancer cells. The expression level of SFMBT2 is high in poorly metastatic prostate cancer cells compared to highly metastatic prostate cancer cells. We also found that SFMBT2 knockdown elevates MMP-2, MMP-3, MMP-9, and MMP-26 expression, leading to increased cell migration and invasion in LNCaP and VCaP cells. SFMBT2 interacts with YY1, RNF2, N-CoR and HDAC1/3, as well as repressive histone marks such as H3K9me2, H4K20me2, and H2AK119Ub which are associated with transcriptional repression. In addition, SFMBT2 knockdown decreased KAI1 gene expression through up-regulation of N-CoR gene expression. Expression of SFMBT2 in prostate cancer was strongly associated with clinicopathological features. Patients having higher Gleason score (≥ 8) had substantially lower SFMBT2 expression than patients with lower Gleason score. Moreover, tail vein or intraprostatic injection of SFMBT2 knockdown LNCaP cells induced metastasis. Taken together, our findings suggest that regulation of SFMBT2 may provide a new therapeutic strategy to control prostate cancer metastasis as well as being a potential biomarker of metastatic prostate cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Signaling regulation and role of filamin A cleavage in Ca2+-stimulated migration of androgen receptor-deficient prostate cancer cells.

Ca2+, a ubiquitous cellular signal, and filamin A, an actin-binding protein, play an important role in the regulation of cell adhesion, shape and motility. Using transwell filters to analyze cell migration, we found that extracellular Ca2+ (Cao2+) promotes the migration of androgen receptor (AR)-deficient and highly metastatic prostate cancer cell lines (DU145 and PC-3) compared to AR-positive and relatively less metastatic prostate cancer cells (LNCaP). Furthermore, we found that expression of filamin A is up-regulated in DU145 and PC-3 cells, and that Cao2+ significantly induces the cleavage of filamin A. Silencing expression of Ca2+-sensing receptor (CaR) and p115RhoGEF, and treating with leupeptin, a protease inhibitor, and ALLM, a calpain specific inhibitor, we further demonstrate that Cao2+-induced filamin A cleavage occurs via a CaR- p115RhoGEF-calpain dependent pathway. Our data show that Cao2+ via CaR- mediated signaling induces filamin A cleavage and promotes the migration in AR-deficient and highly metastatic prostate cancer cells.

A 2-Gene Panel Derived From Prostate Cancer-Enhanced Transcripts in Whole Blood Is Prognostic for Survival and Predicts Treatment Benefit in Metastatic Castration-Resistant Prostate Cancer.

To determine a prognostic model derived from prostate cancer-enhanced transcripts in whole blood of castration-resistant prostate cancer (CRPC) patients and explore its applicability as a surrogate of treatment response. Six out of twenty-three selected transcripts were identified as specific for detection of metastatic prostate cancer cells in peripheral blood using quantitative polymerase chain reaction (qPCR). Their prognostic value was explored in whole blood samples of a training cohort (n = 22 CRPC patients, New York, USA). A resulting 2-gene panel (2GP) including KLK2 and TMPRSS2 was validated in an independent cohort with pre- and post-treatment blood draws after 9-16 weeks of systemic treament (n = 86 CRPC patients, Munich, Germany). Overall survival (OS), prostate-specific antigen progression-free survival (PSA-PFS), and clinical PFS were analyzed. Kaplan-Meier and cox regression analyses were performed. An unfavorable 2GP (≥1 marker positive) identified patients with poor survival (median OS 10.0 months [95%CI 5.7-14.2] vs. not reached; P = 0.023). This was validated in an independent cohort at pre-treatment (median OS 7.8 [95%CI 6.5-9.2] vs. 17.3 months [95%CI 10.7-23.8]; P = 0.004) and post-treatment blood draw (median OS 5.0 [95%CI 0.0-10.0] vs. 18.0 months [95%CI 9.5-26.6]; P = 0.003). The 2GP independently predicted OS on multivariate analysis (hazard ratio 2.1 [95%CI 1.1-4.0]; P = 0.034) and performed better than PSA decline at correlation with OS. Conversion to favorable 2GP during treatment correlated with improved OS (7.8 to 20.9 months), PSA-PFS (2.8 to 12.0 months), and clinical PFS (4.6 to 8.0 months). The established 2GP is prognostic for survival at pre- and post-treatment blood draw in CRPC patients and conversion to favorable 2GP predicts treatment benefit. Prostate 76:1160-1168, 2016. © 2016 Wiley Periodicals, Inc.