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Chemokines and their corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly (p<0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly (p<0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell adhesion to HBME cells and alpha(v)beta(3)-integrin clustering was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.

Background: The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro., Results: Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis., Conclusions: Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.

Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed almost exclusively in prostatic epithelial cells. Expression of PSMA is elevated in prostate cancer, with levels closely correlated with disease grade. Although the highest levels of PSMA expression are associated with high-grade, hormone-refractory and metastatic prostate cancer, the significance of elevated PSMA expression in advanced prostate cancer has yet to be fully elucidated. We provide evidence that prostatic carcinoma cells expressing PSMA exhibit reduced motility and increased attachment when grown on a bone marrow matrix substrate. This phenomenon occurs via activation of focal adhesion kinase and provides the first evidence of a link between PSMA expression and prostate cancer metastasis to the bone.

Background: Stromal cell-derived factor-1 (SDF-1 or CXCL12) and CXCR4 are key elements in the metastasis of prostate cancer cells to bone--but the mechanisms as to how it localizes to the marrow remains unclear., Methods: Prostate cancer cell lines were stimulated with SDF-1 and evaluated for alterations in the expression of adhesion molecules using microarrays, FACs, and Western blotting to identify alpha(v)beta(3) receptors. Cell-cell adhesion and invasion assays were used to verify that activation of the receptor is responsive to SDF-1., Results: We demonstrate that SDF-1 transiently regulates the number and affinity of alpha(v)beta(3) receptors by prostate cancer cells to enhance their metastatic behavior by increasing adhesiveness and invasiveness. SDF-1 transiently increased the expression of beta(3) receptor subunit and increased its phosphorylation in metastatic but not nonmetastatic cells., Conclusions: The transition from a locally invasive phenotype to a metastatic phenotype may be primed by the elevated expression of alpha(v)beta(3) receptors. Activation and increased expression of alpha(v)beta(3) within SDF-1-rich organs may participate in metastatic localization., ((c) 2006 Wiley-Liss, Inc.)

Microvessel density is a prognostic factor for many cancers, including prostate. For this reason, several studies and therapeutic approaches that target the tumor microvasculature have been attempted. Thalidomide has long been recognized as an antiangiogenic molecule. Recently, this drug has regained favor as an anticancer agent and is in clinical trial for multiple myeloma and prostate cancer, among others. This article will briefly review the proposed mechanisms of action for thalidomide, discuss why these activities are of therapeutic value in diseases currently undergoing clinical trials, and summarize the current status of clinical trials for prostate cancer. The focus will be predominantly on the relationship of thalidomide to angiogenesis, as well as on the future and potential value of thalidomide-inspired structural derivatives.

Prostate cancer (PCa) is no exception to the multi-step process of metastasis. As PCa progresses, changes occur within the microenvironments of both the malignant cells and their targeted site of metastasis, enabling the necessary responses that result in successful translocation. The majority of patients with progressing prostate cancers develop skeletal metastases. Despite advancing efforts in early detection and management, there remains no effective, long-term cure for metastatic PCa. Therefore, the elucidation of the mechanism of PCa metastasis and preferential establishment of lesions in bone is an intensive area of investigation that promises to generate new targets for therapeutic intervention. This review will survey what is currently know concerning PCa interaction with the extracellular matrix (ECM) and the roles of factors within the tumor and ECM microenvironments that contribute to metastasis. These will be discussed within the context of changes in expression and functional heterodimerization patterns of integrins, changes in ECM expression and reorganization by proteases facilitating invasion. In this context we also provide a brief summary of how growth factors (GFs), cytokines and regulatory signaling pathways favor PCa metastasis to bone.

Prostate cancer metastasis to bone may be mediated by preferential proliferation of these cells in the bone's microenvironment. We hypothesize that this preferential proliferation is mediated by bone-associated growth factors (GFs) and cytokines. To test our hypothesis, human prostate cancer cells, derived from both soft tissue (LNCaP, DuCaP, DU145) and bone metastases (PC-3, VCaP, MDA-2a, MDA-2b), were treated with bone-associated GFs and cytokines (PDGF, IGF-1, TGF-beta, EGF, bFGF, TNF-alpha, IL-1, and IL-6) for 48 h, and their growth responses were compared. The responses of soft tissue-derived prostate cancer cell lines to bone GFs and cytokines were variable. LNCaP cell growth was stimulated by IGF-1 but was inhibited by TNF-alpha. DU145 cell growth was stimulated with EGF. Prostate cancer cell lines derived from bone metastases also responded variably to bone GFs and cytokines. IL-1 stimulated the growth of MDA-2a and 2b cell lines in a dose-dependent manner. PDGF and bFGF both demonstrated variable effects on bone-derived prostate cancer cell lines. TNF-alpha inhibited proliferation of the VCaP cells. These findings demonstrate that human prostate cancer cell lines derived from bone metastases may not respond preferentially to bone-associated GFs and cytokines.

Background: Prostate carcinoma (PC) frequently metastasizes to bone, where it causes significant morbidity and mortality. Stromal elements in the primary and metastatic target organs are important mediators of tumor cell intravasation, chemoattraction, adhesion to target organ microvascular endothelium, extravasation, and growth at the metastatic site., Methods: The role of stromal factors in bone metastasis was determined with a cyclic DNA microarray comparison of a bone-derived cell PC cell line with a soft tissue-derived cell PC cell line and by evaluating the effects of selected stromal components on PC cell chemotaxis, cell adhesion to human bone marrow endothelium (HBME), and PC cell growth., Results: The authors demonstrate that PC cells express protease-activated receptor 1 (PAR1; thrombin receptor), and its expression is up-regulated in PC compared with normal prostate tissue. In addition, this overexpression was very pronounced in bone-derived PC cell lines (VCaP and PC-3) compared with soft tissue PC cell lines (DUCaP, DU145, and LNCaP). The authors report that bone stromal factors, including stromal cell-derived factor 1 (SDF-1) and collagen Type I peptides, are chemoattractants for PC cells, and they demonstrate that some of these factors (e.g., extracellular matrix components, transforming growth factor beta, bone morphogenic proteins [BMPs], and SDF-1) significantly alter PC-HBME interaction in vitro. Finally, stromal factors, such as BMPs, can regulate the proliferation of PC cells in vitro., Conclusions: Soluble and insoluble elements of the stroma are involved in multiple steps of PC metastasis to bone. The authors hypothesize that PAR1 may play a central role in prostate tumorigenesis., (Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11181)

Objectives: To identify genes important in prostate cancer metastatic to bone. Bone-specific metastasis is a common feature of prostate cancer and a significant cause of morbidity., Methods: To identify factors involved in organ-specific metastasis, we used cDNA microarray analysis to compare a bone-derived cell line, VCaP, with a soft tissue-derived cell line, DuCaP. Both cell lines were derived from the same patient and spontaneously passaged., Results: Forty-five genes were differentially expressed, and only seven of these also had increased expression in VCaP compared with normal prostatic tissue. Of these, protease-activated receptor 1 (PAR1) was verified as having increased expression by reverse transcriptase-polymerase chain reaction and Northern blot analysis, as well as by immunohistochemistry. PAR1 expression in a panel of prostate cancer cell lines demonstrated increased expression in those cell lines derived from bone metastases. Alpha-thrombin stimulation of the VCaP cells produced a dose-dependent mobilization of intracellular calcium compared with DuCaP, suggesting that PAR1 expressed on the VCaP prostate cancer cell line is functional., Conclusions: These data indicate that a functional PAR1 is expressed on prostate cancer cell lines. The prostate cancer cell lines expressing PAR1 appear to have an association with increased bone metastases.

Integrin alpha(v)beta(3) is involved in varied cell biological activities, including angiogenesis, cell adhesion, and migration on several extracellular matrix components. Although alpha(v)beta(3) is not typically expressed in epithelial cells, it is expressed in macrophages, activated leukocytes, cytokine-stimulated endothelial cells, osteoclasts, and certain invasive tumors. Interestingly, the adhesion and migration of breast cancer cells on bone matrix are mediated, in part, by alpha(v)beta(3). Similar to breast cancer cells, prostate cancer cells preferentially metastasize to the bone. The biological events that mediate this metastatic pattern of prostate cancer are not well defined. This review discusses the role alpha(v)beta(3) plays in prostate cancer progression, with specific emphasis on bone metastasis and on alpha(v)beta(3) signaling in prostate cancer cells. The data suggest that alpha(v)beta(3), in part, facilitates prostate cancer metastasis to bone by mediating prostate cancer cell adhesion to and migration on osteopontin and vitronectin, which are common proteins in the bone microenvironment. These biological events require the activation of focal adhesion kinase and the subsequent activation of PI-3 kinase/Akt signaling pathway.

A previous study from our laboratory suggested that prostate cancer metastasis to bone may be mediated, in part, by preferential adhesion to human bone marrow endothelial (HBME) cells. Tumor cell adhesion to endothelial cells may be modulated by the effect of cytokines on cell adhesion molecules (CAMs). Tumor necrosis factor-alpha (TNF-alpha) regulates VCAM expression on the endothelium and this effect is enhanced by dihydrotestosterone (DHT). Transforming growth factor-beta (TGF-beta) stimulates the expression of alpha2beta1 integrin on PC-3 cells. The current study investigated the effects of the above cytokines and DHT (singularly and in various combinations) upon HBME and prostate cancer cell expression of VCAM, alpha2 integrin subunit, and beta1 integrin subunit by flow cytometry. We also monitored the effects of the above treatments on PC-3 cell adhesion to HBME monolayers. The data demonstrate that none of the treatments significantly altered the expression of selected CAMs on HBME cell and neoplastic prostate cell lines. The treatment of HBME monolayers with various combinations of cytokines and DHT prior to performing adhesion assays with PC-3 demonstrates that treatments containing TGF-beta reduced PC-3 cell adhesion to HBME monolayers by 32% or greater (P < 0.05). The reduction in PC-3 cell adhesion to TGF-beta-treated HBME monolayers was dose dependent. Interestingly, LNCaP cells but not PC-3 cells treated with TGF-beta had a reduced ability to adhere to untreated HBME monolayers. These results suggest that TGF-beta may reduce tumor cell adhesion to bone marrow microvascular endothelium, in vivo. The biological significance of this observation is discussed.

The actin cytoskeleton is the key cellular machinery responsible for cellular movement. Changes in the organization and distribution of actin and actin binding protein are necessary for several cellular processes such as focal adhesion formation, cell motility and cell invasion. Here we examined differences in cytoskeletal protein distribution, cell morphometry and cell motility of metastatic and non-metastatic cells. Correlations were found between metastatic potential phenotypic properties such as cell motility, cell spreading and cytoskeletal organization in prostate cancer. As a cell progresses from a normal state to a malignant state, it loses its ability to function normally and also become poorly differentiated. Differentiation therapy is concerned with the redirection of malignant cells toward a terminal, non-dividing state using non-cytotoxic agents. Two well acknowledged differentiation agents, retinoic acid (RA) and diflouromethylomithine (DFMO) were examined for their ability to alter cellular phenotypes associated with metastatic potential in rat prostate cancer cell lines. The results of these studies indicate that there are sub-cellular differences between non-metastatic and highly metastatic cells relative to cytoskeletal organization. We also show that treatment of highly metastatic cells with either RA or DFMO significantly alters cell morphology, cell morphometry and motility to states similar to non-metastatic cells.

The majority of men with progressive prostate cancer develop metastases with the skeleton being the most prevalent metastatic site. Unlike many other tumors that metastasize to bone and form osteolytic lesions, prostate carcinomas form osteoblastic lesions. However, histological evaluation of these lesions reveals the presence of underlying osteoclastic activity. These lesions are painful, resulting in diminished quality of life of the patient. There is emerging evidence that prostate carcinomas establish and thrive in the skeleton due to cross-talk between the bone microenvironment and tumor cells. Bone provides chemotactic factors, adhesion factors, and growth factors that allow the prostate carcinoma cells to target and proliferate in the skeleton. The prostate carcinoma cells reciprocate through production of osteoblastic and osteolytic factors that modulate bone remodeling. The prostate carcinoma-induced osteolysis promotes release of the many growth factors within the bone extracellular matrix thus further enhancing the progression of the metastases. This review focuses on the interaction between the bone and the prostate carcinoma cells that allow for development and progression of prostate carcinoma skeletal metastases.

We have demonstrated previously that the preferential adhesion of prostate cancer cells to human bone marrow endothelial (HBME) cells may contribute to their preferential metastasis to bone. Although a subject of debate, it has been postulated that the endothelial cells of the bone marrow are fenestrated. It is unknown therefore whether prostate cancer cells adhere preferentially to the extracellular matrix (ECM) or the endothelial cells. It has also been demonstrated in other organ systems that the types of cell adhesion molecules on the surface of endothelial cells lining the organ microvasculature are determined, in part, by the ECM of the organ. We investigated how prostate cancer cell adhesion to HBME cells is affected by growing HBME cells on selected organ-derived ECM proteins in vitro. Growth of HBME cells and immortalized human aortic endothelial cells on bone, kidney, and placenta ECM proteins significantly increased their ability to bind PC-3 cells. This increased adhesion was not dose dependent and was not demonstrated with human dermal microvascular endothelial cells. Scanning electron microscopic analysis demonstrated that prostate cancer cells adhered directly to the endothelial cells and not to the underlying substrata. These results suggest that unidentified cell adhesion molecules are expressed or up-regulated on the apical surfaces of human aortic endothelial cells and HBME cells grown on bone, kidney, and placenta ECMs. These results also strongly demonstrate that the adhesion of prostate cancer cells to bone may be initiated by direct binding to endothelial cells rather than direct binding to exposed ECM components.

Background: Previous studies have reported that tumor cells' adhesion to quiescent endothelial cell is mediated by beta-1 integrins. The aim of this study was to determine the role beta-1 integrins play in prostate cancer cell adhesion to human bone marrow endothelial cells (HBME) and human aortic endothelial cells (HAEC)., Materials and Methods: A well described blocking antibody to beta-1 integrin subunit was used in adhesion assays to determine the role of beta-1 integrin subunit in the adhesion of PC-3 cells to both HBME cells and HAEC., Results: Antibody to the beta-1 integrin subunit failed to reduce PC-3 adhesion to HBME and HAEC, yet this same antibody significantly reduced adhesion of PC-3 cells to fibronectin coated wells., Conclusions: The data suggest that metastasis of prostate cancer cells to bone may be mediated, in part, by preferential adhesion to HBME cells; but beta-1 integrins most likely are not involved in this interaction.

Background: Galectin-3 is a carbohydrate-binding protein whose level of expression has been shown to be correlated with metastatic potential in a number of different tumor types. The purpose of this investigation was to examine galectin-3 expression in several tumorigenic and nontumorigenic prostate cell lines and prostate tissue samples., Methods: The expression of galectin-3 in cell lines and tissue samples was evaluated by tissue immunohistochemistry and Western blot analysis., Results: Human cell lines PC-3M, PC-3, DU-145, PrEC-1, and MCF10A demonstrated the presence of galectin-3. Galectin-3 was not detected in TSU-pr1 and LNCaP by Western blot analysis. We furthered our studies by examining a series of human prostate tissue samples for expression of galectin-3. Overall, approximately 60-70% of the normal tissue examined demonstrated heterogenous expression of galectin-3. In stage II tumors, however, there was a dramatic decrease in galectin-3 expression in both PIN and tumor sections, with only 10.5% (2/19) of these samples expressing this protein. Stage III tumors also demonstrated a decreased expression of galectin-3, although this downregulation was not as dramatic, with 35% of PIN samples and 52% of tumor tissue expressing galectin-3 (P < 0.01)., Conclusions: These data demonstrate that galectin-3 is downregulated in prostate cancer. The altered downregulation pattern of galectin-3 observed between tumor stages suggests different roles for galectin-3 in the progression of prostate cancer., (Copyright 2000 Wiley-Liss, Inc.)

Currently, no protocol exists that can assess the metastatic potential of prostate adenocarcinoma. The reason for this is partly due to the lack of information on cellular changes that result in a tumor cell's becoming metastatic. In this investigation, attempts were made to devise a method that correlated with the metastatic potential of AT-1, Mat-Lu, and Mat-LyLu cell lines of the Dunning R-3327 rat prostatic adenocarcinoma system. To accomplish this, we applied BioQuant biometric parameters, i.e., area, shape factor, and cell motility. AT-1 had a lower shape factor and a greater area as compared with the more highly metastatic Mat-Lu subline. No significant difference in area or shape factor was detected between the AT-1 cell line and the highly metastatic Mat-LyLu line. However, the lowly metastatic AT-1 line had less motility as compared with the Mat-Lu and Mat-LyLu lines. This study revealed that metastatic potential could be partially predicted via area and shape factor and accurately predicted via cell motility.

This investigation examined the ability of low-metastatic AT-1 and high-metastatic ML and MLL cell lines to adhere to a monolayer of bovine aortic endothelial cells and fibronectin substratum and penetrate a matrigel basement membrane matrix. Cell area, shape factor, and motility as influenced by fibronectin were also examined using these cell lines. The ability of these cells to adhere to fibronectin and traverse matrigel matrix correlated with their metastatic potential. In addition, fibronectin increases cell circularity and reduces cell area as metastatic potential increases within AT-1 to ML or MLL cell lineages.