CXCR6 is expressed in human prostate cancer in vivo and is involved in the in vitro invasion of PC3 and LNCap cells

In spite of the clinical importance of prostate cancer (PCa) bone metastasis, the precise mechanisms for the directed migration of malignant cells remain unclear. In the present study, the expression of CXCR6 in human PCa and benign prostatic hyperplasia samples, and the expression of CXCL16 in human osseous tissues were determined by immunohistochemistry. It was found that the level of CXCR6 protein expression was elevated in human malignant prostate tumors, and CXCL16 was expressed positively by human osteocytes in vivo . The in vitro experiments further confirmed that the PCa cell lines PC3 and LNCap expressed CXCR6 at both the mRNA and protein levels, and exogenous CXCL16 has the potential to stimulate the invasion of PC3 and LNCap. To further elucidate the role of the CXCL16–CXCR6 axis in PCa progression, we compared the expression of CXCR6 and CXCR4 in human PCa tissues and the effects of CXCL16 and CXCL12 on the in vitro invasion of PC3 and LNCap cells. It was shown that CXCR6 and CXCR4 proteins were coexpressed and elevated in human PCa samples, and CXCL16 and CXCL12 promoted the invasion of PC3 and LNCap via their respective receptors. Furthermore, in contrast to CXCL12, which enhanced the activity of matrix metalloproteinase (MMP) 9 and MMP2 in PC3 and LNCap, CXCL16 ligation resulted in stronger MMP9 and MMP2 activity in LNCap but not in PC3. Our results suggest that besides CXCL12/CXCR4, CXCL16/CXCR6 might be another important factor involved in PCa bone metastasis. ( Cancer Sci 2008; 99: 1362–1369)     

Basic fibroblast growth factor in human prostate cancer cells.

To increase our understanding of the potential role of basic fibroblast growth factor (bFGF) in malignant progression of prostate cancer, we determined the production of bFGF, the expression of FGF receptor (flg), and the response to exogenous bFGF in LNCaP, DU 145, and PC 3 cells. We observed that these three prostate cancer cell lines, which differed in their dependence on androgens for growth in vitro and in their in vivo behavior in nude mice, could be distinguished as follows: (a) androgen-sensitive LNCaP cells, which do not metastasize in nude mice, did not produce measurable amounts of bFGF, expressed small but measurable amounts of FGF receptor mRNA, and did respond to exogeneous bFGF; (b) androgen-insensitive, moderately metastatic DU 145 cells did produce measurable amounts of biologically active bFGF, expressed large amounts of FGF receptor mRNA, and responded to exogeneous bFGF and the heparin-binding fractions from DU 145 cell extracts; (c) androgen-insensitive and highly metastatic PC3 cells also produced measurable amounts of bFGF but did not demonstrate a growth response to either the heparin-binding fractions from PC3 cell extracts or exogenous bFGF, even though large amounts of FGF receptor mRNA were expressed in PC 3 cells. These results suggest the possibility that differences in production of, and response to, bFGF may be associated with different biological behavior.     

CXCR4-mediated adhesion and MMP-9 secretion in head and neck squamous cell carcinoma

The chemokine CXCL12 (SDF-1) and its receptor, CXCR4, have been implicated in organ-specific metastases of several malignancies. Head and neck squamous cell carcinoma (HNSCC) predominantly metastasizes to lymph nodes, and recent evidence has shown that CXCL12 stimulates HNSCC migration. We explored the potential role of CXCR4 in mediating other metastatic processes in HNSCC cells. CXCR4 mRNA and cell-surface expression was assessed in HNSCC cell lines. CXCR4 mRNA expression was detected in five HNSCC cell lines. Cell-surface CXCR4 was also detected in each of the HNSCC cell lines and in resected HNSCC tissues. CXCL12 induced rapid intracellular calcium mobilization in a metastatic HNSCC cell line (HN), as well as rapid phosphorylation of ERK-1/2. HNSCC cell adhesion to fibronectin and collagen was increased by CXCL12 treatment, while the addition of an inhibitor of ERK-1/2 signaling, PD98059, reduced the effects of CXCL12. CXCL12 also increased the active matrix metalloproteinase (MMP)-9 secreted. Thus, HNSCC cells express functional CXCR4 receptors that induce rapid intracellular signaling upon binding to CXCL12. Such binding leads to increased HNSCC cell adhesion and MMP secretion, suggesting that CXCR4 may be a novel regulator of HNSCC metastatic processes.     

Prostate cancer mediates osteoclastogenesis through two different pathways

The present study was undertaken to test the effects of prostate cancer cell lines (LNCaP, DU145, PC3, and MDA PCa 2b) on osteoclastogenesis. Crude conditioned medium (CM) from all four prostate cancer cell lines enhanced expression of the mRNA for receptor activator of NF-kappaB ligand (RANKL) in a mouse osteoblast cell line, MC3T3-E1; however, CM had no effect on expression of osteoprotegerin (OPG) mRNA. Coculture of MC3T3-E1 with prostate cancer cells yielded similar results. The number of mature osteoclasts induced by soluble RANKL increased significantly when osteoclast precursor cells were cultured with CM from LNCaP and DU145 cells. CM from LNCaP and DU145 cells also induced maturation from precursor in the absence of soluble RANKL, and this effect was not blocked by OPG. Addition of CM from DU145 cells increased expression of MMP-9 mRNA by osteoclast precursors. Our findings indicate that prostate cancer mediates osteoclastogenesis through induction of RANKL expression by osteoblasts and through direct actions on osteoclast precursors mediated by some factors other than RANKL.     

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.     

Altered response to thyroid hormones by prostate and breast cancer cells

Transferrin, an abundant bone marrow constituent, has been shown to be a potent mitogen in vitro in the prostate cancer cell line PC3. T4 (L-thyroxine) and T3 (3′,3,5-tri-iodo-L-thyronine) are regulators of cell metabolism. In this study, the effects of nonphysiological concentrations (about two orders of magnitude higher) of T4, T3, T2 (3,5-di-iodo-L-thyronine), RT3 (reverse T3, 3′,5′,3-tri-iodo-L-thyronine) and transferrin (about three orders of magnitude lower) were tested on the prostate cancer cell lines PC3, DU145 and LNCaP, and the breast cancer cell line MCF-7. In PC3 cells, increased proliferation by transferrin could be reversed by the addition of T3 or T4. T4 decreased proliferation in all cell lines tested, while transferrin increased proliferation in PC3 cells only. T3 decreased proliferation in PC3, LNCaP and MCF-7 cells but had no effect on DU145 cells. T4 and T3 gave two-state behavior in LNCaP cells. These results were combined to determine the essential iodines which produced the observed proliferative effects. Cell lines responded differently to T4, T3, T2, RT3 and transferrin suggesting a specific interaction among the compounds tested and the different cell lines. Finally, regulation of gene expression was demonstrated using DU145 cells. Upregulation of c-fos mRNA was observed in cultures at early time-points in the presence of T4, transferrin or both. Decreased expression was observed at later time-points with no expression at 4 h. An explanation for these results may be a change in thyroid hormone receptor/ligand affinity. Thus, the interactions between thyroid hormones and cancer cells may be different from those between thyroid hormones and normal cells. Received: 28 January 1999 / Accepted: 26 August 1999     

Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells

Focal adhesion kinase (FAK) has been implicated in the regulation of cell migration. In addition, FAK expression is increased in a number of highly metastatic tumor cell lines. Therefore, we investigated the role of FAK in regulating migration of prostate carcinoma cell lines with increasing metastatic potential. We show that highly tumorigenic PC3 and DU145 cells exhibit intrinsic migratory capacity, while poorly tumorigenic LNCaP cells require a stimulus to migrate. Increased metastatic potential of PC3 and DU145 cells correlates with increased FAK expression, overall tyrosine phosphorylation and activity, as measured by autophosphorylation of tyrosine 397. However, in PC3 and DU145 cells, FAK autophosphorylation is adhesion dependent whereas a second site of tyrosine phosphorylation, tyrosine 861, a Src specific site, is uncoupled from adhesion-dependent signaling events. Finally, inhibiting the FAK/Src signal transduction pathway by over expressing FRNK (Focal adhesion kinase-Related Non-Kinase), an inhibitor of FAK activation, or treatment with PP2, a Src family kinase inhibitor, significantly inhibited migration of prostate carcinoma cell lines, demonstrating that tumor cell migration continues to be dependent on signals emanating from this pathway.     

Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell-cycle variation during in vitro differentiation.

The CXCL12 / CXCR4 system may be important in carcinoma. Expression of the alpha-chemokine SDF-1alpha (stromal cell derived factor-1alpha) / CXCL12 mRNA is reduced in many carcinomas, yet its tissue protein expression may guide metastasis. Here we first compare the mRNA and protein expression of CXCL12 and its receptor CXCR4 in human liver, hepatocellular carcinoma, and malignant cell lines, and then assess cell cycle variation in CXCR4 expression. CXCR4 mRNA was present in most normal human tissues and malignant cell lines; it was only marginally reduced in hepatomas, while CXCL12 was markedly reduced, P < 0.0001. Immuno-histochemical staining of adjacent non-malignant liver showed regional CXCR4 cytoplasmic and cell-surface staining, limited to those hepatocytes around the central vein, a distribution resembling that of CXCL12. CXCL12 protein was not present in hepatocellular carcinoma cells in vivo, nor was cytoplasmic CXCR4 staining; nuclear CXCR4 protein expression in some malignant hepatocytes and CXCR4 staining of capillary endothelial cells around tumor cells were noted. In some malignant cell lines that had no CXCL12 on northern blots CXCL12 was weakly detectable by RT-PCR or protein staining in the cytoplasm of a few cells. With a view to future manipulation of CXCL12 / CXCR4 expression and growth we noted that in HT-29 cells CXCR4 protein expression was less on confluent than on non-confluent cells and varied during the cell cycle. Higher expression was associated most closely with the percentage of cells in the S-phase and inversely with the percentage of cells in the G1-phase. Treatment of HT-29 cells with butyrate reduced CXCR4 cell surface expression and reduced the percentage of cells in S-phase. In summary, CXCL12 protein expression parallels its mRNA, being markedly reduced in malignant cell lines and hepatomas; in liver, the regional distributions of CXCL12 and cytoplasmic CXCR4 are similar; finally, in HT-29, CXCR4 expression correlates with the S-phase of the cell cycle and is reduced during butyrate-induced differentiation.     

Clinical and biological significance of CXCR5 expressed by prostate cancer specimens and cell lines

Chemokines and chemokine receptors have been shown to be involved in metastatic process of prostate cancer (PCa). In this study, we show primary PCa tissues and cell lines (LNCaP and PC3) express CXCR5, a specific chemokine receptor for CXCL13. Expression of CXCR5 was significantly higher (p < 0.001) in PCa cases than compared to normal match (NM) tissues. CXCR5 intensity correlated (R² = 0.97) with Gleason score. While prostate tumor tissues with Gleason scores ≥ 7, displayed predominantly nuclear CXCR5 expression patterns, PCa specimens with Gleason scores ≤ 6 showed predominantly membrane and cytoplasmic expression patterns that were comparable to benign prostatic hyperplasia (BPH). Similar to tissue expression, PCa cell lines expressed significantly more CXCR5 than normal prostatic epithelial cells (PrECs), and CXCR5 expression was distributed among intracellular and extracellular compartments. Functional in vitro assays showed higher migratory and invasive potentials toward CXCL13, an effect that was mediated by CXCR5. In both PCa cell lines, CXCL13 treatment increased the expression of collagenase‐1 or matrix metalloproteinase‐1 (MMP‐1), collagenase‐3 (MMP‐13), stromelysin‐1 (MMP‐3), stromelysin‐2 (MMP‐10) and stromelysin‐3 (MMP‐11). These data demonstrate the clinical and biological relevance of the CXCL13‐CXCR5 pathway and its role in PCa cell invasion and migration. © 2009 UICC     

CXCR4 Inhibition with AMD3100 Sensitizes Prostate Cancer to Docetaxel Chemotherapy

Several in vitro and in vivo models have revealed the key role of CXCR4/CXCL12 axis in tumor-stroma interactions. Stromal cells present in the tumor microenvironment express high levels of CXCL12 protein, directly stimulating proliferation and migration of CXCR4-expressing cancer cells. This specific prosurvival influence of stromal cells on tumor cells is thought to protect them from cytotoxic chemotherapy and is postulated as a possible explanation for the minimal residual disease in hematological and solid cancers. Therefore, CXCR4/CXCL12 signaling is an attractive therapeutic target in cancer, as proven in preclinical leukemia mouse models, where CXCR4 inhibition sensitized cancer cells to conventional chemotherapy. This study investigates whether inhibition of CXCR4 with the specific inhibitor AMD3100 sensitizes human prostate cancer cells to docetaxel. We showed that both mouse and human stromal cell lines have a protective effect on PC3-luc cells by promoting their survival after chemotherapy. Furthermore, we demonstrated that AMD3100 sensitizes PC3-luc cells to docetaxel. In a subcutaneous xenograft mouse model of human prostate carcinoma, we showed that a combination of docetaxel and AMD3100 exerts increased antitumor effect compared with docetaxel alone. We concluded that CXCR4 inhibition chemosensitizes prostate cancer cells, both in vitro and in vivo. To explore the relevance of these findings, we analyzed CXCR4 expression levels in human prostate cancer samples. We found that cancer cells present in bone metastatic lesions express higher CXCR4 levels relative to the cells present in primary tumors and lymph node metastatic lesions. These findings underscore the potential of CXCR4 inhibitors as chemosensitizing agents.     

CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 +ve) and DU145 (CD44 -ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 +ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.     

Imaging agents for in vivo molecular profiling of disseminated prostate cancer--targeting EGFR receptors in prostate cancer: comparison of cellular processing of [111In]-labeled affibody molecule Z(EGFR:2377) and cetuximab

Expression of receptor tyrosine-kinase (RTK) EGFR is low in normal prostate, but increases in prostate cancer. This receptor is significantly up-regulated as tumors progress into higher grade, androgen-insensitive and metastatic lesions. The up-regulated receptors could serve as targets for novel selective anti-cancer drugs, e.g. antibodies and tyrosine kinase inhibitors. Radionuclide imaging of RTK can facilitate patient stratification and monitoring of anti-RTK therapy of prostate cancer. The goal of the study was to evaluate binding and cellar processing of radiolabeled EGFR-targeting conjugates by prostate cancer cell lines. Receptor expression of EGFR was studied in three prostate cancer cell lines: DU145 (brain metastasis of PC, hormone insensitive), PC3 (bone metastasis of PC) and LNCaP (lymph node metastasis of PC, androgen and estrogen receptor positive). Uptake and internalization of anti-EGFR mAbs (cetuximab) and affibody molecule (Z2377) labeled with indium-111 was investigated. EGFR expression on prostate cancer cell lines was clearly demonstrated. Both labelled conjugates 111In-Z2377 and 111In-cetuximab bound to prostate cancer cells in the receptor mediated model. Expression levels were modest but correlate with degree of hormone independence. Internalization of Affibody molecules was relatively slow in all cell lines. Internalization of mAbs was more rapid. The level of EGFR expression in these cell lines is sufficient for in vivo molecular imaging. Slow internalization indicates possibility of the use of non-residualizing labels for affibody molecules.     

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer

Protectin (CD59) inhibits homologous complement-mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18-25 kDa. Treatment of DU145 and PC3 cells with phosphatidylinositol-specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement-mediated cytolysis but became sensitive to killing in the presence of the CD59-neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI-linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement. Int. J. Cancer 71: 1049-1055, 1997.© 1997 Wiley-Liss Inc.