Bone Metastatic Prostate Cancer Cells Research Articles

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.

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.

Inhibition of skeletal growth of human prostate cancer by the combination of docetaxel and BKM1644: an aminobisphosphonate derivative.

Bone metastasis is a major cause of prostate cancer (PCa) morbidity and mortality. Despite some success in transiently controlling clinical symptoms with docetaxel-based therapy, PCa patients become docetaxel-resistant and inevitably progress with no cure. We synthesized an acyl-tyrosine bisphosphonate amide derivative, BKM1644, with the intent of targeting bone metastatic PCa and enhancing docetaxel's efficacy. BKM1644 exhibits potent anti-cancer activity in the NCI-60 panel and effectively inhibits the proliferation of metastatic, castration-resistant PCa (mCRPC) cells, with IC50 ranging between 2.1 μM and 6.3 μM. Significantly, BKM1644 sensitizes mCRPC cells to docetaxel treatment. Mice with pre-established C4-2 tumors in the tibia show a marked decrease in serum prostate-specific antigen (control: 173.72 ± 37.52 ng/ml, combined treatment: 64.45 ± 22.19 ng/ml; p < 0.0001) and much improved bone architecture after treatment with the combined regimen. Mechanistic studies found that docetaxel temporarily but significantly increases survivin, an anti-apoptotic protein whose overexpression has been correlated with PCa bone metastasis and therapeutic resistance. Intriguingly, BKM1644 effectively inhibits survivin expression, which may antagonize docetaxel-induced survivin in bone metastatic PCa cells. Signal transducer and activator of transcription 3 (Stat3) may be involved in the suppression of survivin transcription by BKM1644, as confirmed by a survivin reporter assay. Collectively, these data indicate that BKM1644 could be a promising small-molecule agent to improve docetaxel efficacy and retard the bone metastatic growth of PCa.

MiR-154* and miR-379 in the DLK1-DIO3 microRNA mega-cluster regulate epithelial to mesenchymal transition and bone metastasis of prostate cancer.

MicroRNAs in the delta-like 1 homolog-deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature (“Core” OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Abstract 2030: Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies

Abstract Advanced prostate cancer (PCa) typically metastasizes to bone and current treatments are mainly palliative. While a mechanistic understanding of PCa progression in bone is needed to develop new therapies, the complex phenotypes in PCa are poorly reflected by cell lines and cell line-based xenograft models, impeding progress in this endeavor. To overcome this limitation, a panel of PCa patient-derived xenograft (PDX) models was established at the University of Texas MD Anderson Cancer Center. While these PDX models better recapitulate the phenotypic signature of the original tumors as compared to cell lines, limitations exist, including the inability to manipulate these cells ex vivo before implantation in vivo and issues associated with the foreign animal physiology. Based on previous studies demonstrating the potential of using hyaluronan (HA)-based hydrogels for the 3D engineering of PCa cell line-derived ectotumors suitable for mechanistic studies, we evaluated the ability of these hydrogels to maintain viability of the PCa PDX tumors in vitro and optimize culture conditions. Using the MDA PCa 183 and 118b PDX tumors as proof-of-concept, tumors freshly harvested were digested and encapsulated within a HA-based hydrogel, comprised of thiol-modified HA crosslinked with poly(ethylene glycol)-diacrylate. Tumor cells remained as multicellular aggregates or ectotumors in 3D, and were viable even up to 2 weeks in culture. Furthermore, cells were positive for Ki-67, indicating that the hydrogel system is able to retain the proliferative capacity of the tumor cells. In probing for the androgen receptor (AR) expression, we found that while the hydrogel-encapsulated MDA PCa 183 cells stained positive for AR, it was not the case for the MDA PCa 118b, as would be expected in vivo, demonstrating that the hydrogel system is also able to preserve critical phenotypic characteristics of the original tumors. Lastly, to evaluate this novel system as a potential platform for rapid drug testing, we treated the 3D PDX ectotumors to docetaxel, and compared their response against a traditional bone metastatic PCa cell line, C4-2B. Interestingly, the 3D PDX ectotumors exhibited a significantly greater resistance to the drug than the C4-2B cells, reminiscent of chemoresistant tumors in vivo. We demonstrate for the first time that PCa PDX tumors are amenable to in vitro culture with the use of a 3D HA-based hydrogel system. The hydrogel supported the maintenance of multicellular ectotumors of PCa cells, which remained viable and phenotypically similar to the tumors of origin. Furthermore, a differential drug response to docetaxel was observed for both the 3D PCa PDX ectotumors tested as compared to the C4-2B cell line, the clinical relevance of which is currently being investigated. This model may have broad applicability for the culture of other bone metastatic cancers and enable mechanistic studies of disease biology. Citation Format: Eliza L.S. Fong, Mariane Martinez, Jun Yang, Leland M.K. Chung, Antonios G. Mikos, Nora M. Navone, Daniel A. Harrington, Mary C. Farach-Carson. Culturing patient-derived prostate cancer xenografts using a three-dimensional hydrogel system for drug testing and mechanistic studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2030. doi:10.1158/1538-7445.AM2014-2030

MiR-409-3p/-5p promotes tumorigenesis, epithelial-to-mesenchymal transition, and bone metastasis of human prostate cancer.

miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle-treated cells. miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer.

Discoidin domain receptor 2 facilitates prostate cancer bone metastasis via regulating parathyroid hormone-related protein

Prostate cancer frequently metastasizes to the skeleton but the underlying mechanism remains largely undefined. Discoidin domain receptor 2 (DDR2) is a member of receptor tyrosine kinase (RTK) family and is activated by collagen binding. This study aimed to investigate the function and detailed mechanism of DDR2 in prostate cancer bone dissemination. Herein we found that DDR2 was strongly expressed in bone-metastatic prostate cancer cells and tissues compared to that in normal controls. Enhanced expression of constitutively activated DDR2 led to elevation in motility and invasiveness of prostate cancer cells, whereas knockdown of DDR2 through specific shRNA caused a dramatic repression. Knockdown of DDR2 in prostate cancer cells resulted in significant decrease in the proliferation, differentiation and function of osteoblast. Over-expression of DDR2 in prostate cancer cells resulted in notable acceleration of osteoclast differentiation and bone resorption, whereas knockdown of DDR2 exhibited the opposite effects. An intrabone injection bone metastasis animal model demonstrated that DDR2 promoted osteolytic metastasis in vivo. Molecular evidence demonstrated that DDR2 regulated the expression, secretion, and promoter activity of parathyroid hormone-related protein (PTHrP), via modulating Runx2 phosphorylation and transactivity. DDR2 was responsive to TGF-β and involved in TGF-β-mediated osteoclast activation and bone resorption. In addition, DDR2 facilitated prostate cancer cells adhere to type I collagen. This study reveals for the first time that DDR2 plays an essential role in prostate cancer bone metastasis. The mechanism disclosure may provide therapeutic targets for the treatment of prostate cancer.

Anterior gradient protein-2 is a regulator of cellular adhesion in prostate cancer.

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

Induction of integrin α2 in a highly bone metastatic human prostate cancer cell line: roles of RANKL and AR under three-dimensional suspension culture.

BackgroundProstate cancer (PCa) bone metastasis can be markedly enhanced by increased receptor activator of NF kappa-B ligand (RANKL) expression in PCa cells. Molecular mechanisms that account for the increased predilection of PCa for bone include increased bone turnover, promotion of PCa cell growth and survival in the bone environment, and recruitment of bystander dormant cells to participate in bone metastasis. The current study tests the hypothesis that PCa cells acquire high adhesion to bone matrix proteins, which controls PCa bone colonization, under the RANKL/RANK and AR axes.MethodsWe used a highly bone metastatic RANKL-overexpressing LNCaP PCa cell line, LNCaPRANKL, as a model to pursue the molecular mechanisms underlying the increased adhesion of PCa cells to collagens. A three-dimensional (3-D) suspension PCa organoid model was developed. The functions of integrin α2 in cell adhesion and survival were evaluated by flow cytometry and western blot. AR expression and functionality were compared in 2-D monolayer versus 3-D suspension cultures using AR promoter- and PSA promoter-luciferase activity. AR role in cell adhesion was assessed using an adhesion assay.ResultsLNCaPRANKL cells were shown to adhere tightly to ColI matrix through increased α2 integrin expression. This increased adhesion, concomitant with activation of the FAK and Akt pathways, was further enhanced by culturing LNCaPRANKL cells in 3-D suspension. Under the influence of 3-D suspension culture, AR was restored in LNCaPRANKL cells via downregulation of AP-4 transcription factor, and supported increased α2 integrin expression and adhesion to ColI.Conclusion3-D suspension culture and in vivo PCa tumor growth restore AR through downregulation of AP-4, enhancing integrin α2 expression and adhesion to ColI which is rich in bone matrices. The interactions of PCa with ColI, mediated by integrin α2 and AR expression, could be a key molecular event accounting for PCa bone metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-208) contains supplementary material, which is available to authorized users.

P62/SQSTM1 is required for cell survival of apoptosis‐resistant bone metastatic prostate cancer cell lines

Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.

Interleukin-1β Promotes Skeletal Colonization and Progression of Metastatic Prostate Cancer Cells with Neuroendocrine Features

Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1β (IL-1β), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1β by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1β in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1β secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1β and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1β supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.

Bone metastatic prostate cancer cells regulate their growth via impairing osteoblast differentiation

Bone metastatic prostate cancer cells regulate their growth via impairing osteoblast differentiation

Abstract 4997: Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer.

Abstract Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar W. Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4997. doi:10.1158/1538-7445.AM2013-4997

Abstract B11: Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer

Abstract Introduction and Objective: While prostate cancer (PCa) is curable at early diagnosis, metastatic PCa is terminal. Recent studies have shown that Receptor Activator of NF kappa-B Ligand, RANKL, markedly enhances bone metastasis (70-100%) by the LNCaP human PCa cell line in mice compared to neo controls (0%). Multivariable tumor and microenvironmental factors in vivo make it difficult to identify the specific cellular response induced by RANKL causing PCa cell homing and growth in bone. Current in vitro 2D cultures lack mechanical and biochemical properties mimicking the bone microenvironment. Our preliminary data showed integrin alpha2 is elevated in RANKL-over-expressing LNCaP cells. PCa interaction with the major bone matrix protein Col1 may contribute to the metastatic potential of LNCAP-RANKL cells. This study used 3D systems to explore the role of RANKL in PCa-bone matrix interactions. Methods: 3D cultures of RANKL-expressing (highly metastatic) or non-metastatic LNCaP cells were evaluated in 2D, suspension cultures, on type 1 collagen (Col1) coated polymeric meshes, embedded in Col1 gel or hydrogel mixed with Col1 for growth, morphology, motility, and gene expression. Time-dependent cell migration was assessed by a time-lapse imaging system. Results: LNCAP-RANKL cells and Neo-expressing controls grew as aggregates in all 3D cultures but not 2D cultures. Col1 promoted less compact and more widespread aggregation of LNCAP-RANKL cells in the 3D cultures, exhibiting more motile and directional migratory behavior. A molecular signature of 3D growth was generated and compared among different conditions. Consistent with 2D studies, 3D qRT-PCR data showed RANKL drove epithelial-to-mesenchymal transition (EMT), shown by increased expression of vimentin and N-cadherin but decreased E-cadherin. Decreased expression of androgen receptor and PSA was observed in RANKL-expressing PCa cells. Supporting the migration data, the presence of Col1 drove RANKL-expressing PCa cells to express higher levels of c-Met protooncogene, sensitizing PCa cell responses to the c-Met ligand, hepatocyte growth factor (HGF). Conclusions: 3D culture systems were established to study interactions between Col1 and metastatic or non-metastatic PCa cells. 3D culture systems supported PCa cell growth, while maintaining their classical markers, validating the reliability of our 3D systems. Bone metastatic PCa cells preferentially exhibited EMT, increased expression of α2 integrin, and c-Met protooncogene. These 3D systems will be used to assess liquid biopsy specimens harvested from human PCa patients to improve prognosis and therapeutic follow-up. Citation Format: Shabnam Ziaee, Shirly Sieh, Chia-Yi Chu, Ruoxiang Wang, Dietmar Hutmacher, Colleen Nelson, Chin-Lin Guo, Leland W.K. Chung. Using three-dimensional (3-D) culture systems to delineate the role of RANKL in metastatic prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B11.

In vitro the behaviors of metastasis with suppression of VEGF in human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line.

BackgroundVascular endothelial growth factor (VEGF) is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis. VEGF is believed to implicate poor prognosis in various cancers. The overexpression of VEGF may be an early step in the process of metastasis.MethodsELISA was used to investigate the levels of VEGF, bFGF and IL8 in human bone metastatic LNCaP-derivative C4-2B prostate cancer cell line and its parental cell line, LNCaP and to determine the effect of bevacizumab on reducing the level of VEGF. Cell proliferation assay, invasion assay and in vitro angiogenesis assay were performed under the condition with bevacizumab or control IgG.ResultsHuman bone metastatic LNCaP-derivative C4-2B prostate cancer cell line expressed a higher level of VEGF than its parental primary prostate cancer cell line LNCaP. The effect of bevacizumab is dose-dependent and time-dependent: 100 μg/mL of bevacizumab and 3-day treatment was more effective than low-dose and lesser-day treatment for decreasing the level of VEGF. Bevacizumab is able to suppress cell proliferation, angiogenesis and invasion in human bone metastatic C4-2B prostatic cancer cell line.ConclusionsThe overexpression of VEGF can be inhibited by bevacizumab in human bone metastatic cancer cell line. The behaviors of metastasis involving proliferation, angiogenesis and invasion are suppressed by anti-VEGF therapy.

Osteoblast differentiation stage determines the bidirectional cross-talk with bone metastatic prostate cancer cells

Osteoblast differentiation stage determines the bidirectional cross-talk with bone metastatic prostate cancer cells

Abstract 3206: Three-dimensional hyaluronan hydrogel culture systems facilitate study of the complex microenvironment of bone metastatic prostate cancer cells

Abstract The bone marrow microenvironment is a complex milieu, containing various stromal and endothelial cell populations as well as a unique extracellular matrix. To better understand cellular interactions that occur in potentially lethal stage IV prostate cancer (PCa), it is helpful to recreate the bone marrow microenvironment. Traditional two dimensional (2D) cell culture systems do not accurately portray this environment, since culture on plastic activates atypical signaling pathways and makes co-culture of various cell types difficult. To overcome these limitations, we developed novel methods of three-dimensional (3D) culture to study the biology of bone metastatic PCa cells. These 3D culture systems utilize ECM-modified hyaluronan (HA) hydrogels to culture PCa cells and bone marrow stromal cells (BMSC) in a variety of well controlled configurations. The HA hydrogel reflects the low modulus environment of the bone marrow as well as HA's prevalence within the tissue. Various types of PCa cells, including cells derived from patient bone metastases, were cultured in HA hydrogels and viability was maintained as assessed by live/dead assays over time. Because HA is a bio-active signaling molecule that can interact with PCa cells through its receptors, cluster of differentiation 44 (CD44) or receptor for hyaluronan mediated motility (RHAMM), changes in morphology and signaling pathway activation were observed in response to the HA hydrogel culture. To investigate how PCa and BMSC interact, we encapsulated BMSC (HS27a) within a collagen-modified HA hydrogel. These cells formed a reticular network in the 3D matrix, reminiscent of the architecture of bone marrow stroma. When co-cultured with BMSC, bone metastatic PCa cells adhered to the reticular network of stromal cells and exhibited phenotypic changes suggesting neuroendocrine differentiation. We are developing a bilayered HA-based system to model, in 3D, the interface between PCa and the bone marrow stroma. Our model system includes 6 mm diameter disks of HA composed of two well-defined layers, one encapsulating BMSCs and the other containing PCa cells; each cell line can be labeled with a different fluorescent protein. These bilayered disks can be imaged to study the means of invasion of PCa into bone marrow stroma mimicking metastasis. Changes in cell-cell interactions and in cell morphologies in 3D can be imaged without disturbing the dynamic system, allowing for experiments to be monitored over time. These structured 3D units provide a novel means to study cancer and stroma invasiveness, cell-cell interactions and phenotypic changes in a 3D biomimetic system that simulates the bone microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3206. doi:1538-7445.AM2012-3206

Abstract 5173: The human homologue of frog (Xenopus laevis) anterior gradient protein promotes metastasis via regulation of cellular adhesion

Abstract Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV &amp; V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5173. doi:1538-7445.AM2012-5173

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.